Multiparameter FACS Assays to Detect Alterations in Cellular Parameters and to Screen Small Molecule Libraries

ABSTRACT

The invention relates to novel methods of detecting alterations in cell cycle regulation in a cell or a cell population and screening for agents capable of modulating cell cycle regulation through the use of multiparameter assays and a fluorescence-activated cell sorter (FACS) machine.

FIELD OF THE INVENTION

The invention relates to novel methods of detecting alterations incellular parameters, and particularly for screening libraries of smallmolecules such as combinatorial chemical libraries of organic molecules,including peptides and other chemical libraries, for binding to targetmolecules, using fluoroscence-activated cell sorting (FACS) machines.

BACKGROUND OF THE INVENTION

The field of drug discovery and screening of drug candidates to identifylead compounds is rapidly expanding. Traditional approaches to identifyand characterize new and useful drug candidates include the isolation ofnatural products or synthetic preparation, followed by testing againsteither known or unknown targets. See for example WO 94/24314, Gallop etal., J. Med. Chem. 37(9):1233 (1994); Gallop et al., J. Med. Chem.37(10):1385 (1994); Ellman, Acc. Chem. Res. 29:132 (1996); Gordon etal., E. J. Med. Chem. 30:388s (1994); Gordon et al., Acc. Chem. Res.29:144 (1996); WO 95/12608, all of which are incorporated by reference.

The screening of these libraries is done in a variety of ways. Oneapproach involves attachment to beads and visualization with dyes; seeNeslter et al., Bioorg. Med. Chem. Lett. 6(12):1327 (1996). Anotherapproach has utilized beads and fluorescence activated cell sorting(FACS); see Needles et al., PNAS USA 90:10700 (1993), and Vetter et al.,Bioconjugate Chem. 6:319 (1995).

Fluorescence activated cell sorting (FACS), also called flow cytometry,is used to sort individual cells on the basis of optical properties,including fluorescence. It is generally fast, and can result inscreening large populations of cells in a relatively short period oftime.

There are a number of instances where rapid and inexpensive screens suchas FACS screens would be of particular interest. On such area is in cellcycle assays. Cells cycle through various stages of growth, startingwith the M phase, where mitosis and cytoplasmic division (cytokinesis)occurs. The M phase is followed by the G1 phase, in which the cellsresume a high rate of biosynthesis and growth. The S phase begins withDNA synthesis, and ends when the DNA content of the nucleus has doubled.The cell then enters G2 phase, which ends when mitosis starts, signaledby the appearance of condensed chromosomes. Terminally differentiatedcells are arrested in the G1 phase, and no longer undergo cell division.

The hallmark of a malignant cell is uncontrolled proliferation. Thisphenotype is acquired through the accumulation of gene mutations, themajority of which promote passage through the cell cycle. Cancer cellsignore growth regulatory signals and remain committed to cell division.Classic oncogenes, such as ras, lead to inappropriate transition from G1to S phase of the cell cycle, mimicking proliferative extracellularsignals. Cell cycle checkpoint controls ensure faithful replication andsegregation of the genome. The loss of cell cycle checkpoint controlresults in genomic instability, greatly accelerating the accumulation ofmutations which drive malignant transformation. Hence, checkpointregulators, such as p53 and ATM (ataxia telangiectasia mutated), alsofunction as tumor suppressors. Thus, modulating cell cycle checkpointpathways with therapeutic agents could exploit the differences betweennormal and tumor cells, both improving the selectivity of radio- andchemotherapy, and leading to novel cancer treatments.

Accordingly, it is an object of the invention to provide compositionsand methods useful in screening for modulators of cell cycle checkpointregulation.

Another area for which rapid screening methods would find particular useis in the area of assays of exocytosis. Exocytosis is the fusion ofsecretory vesicles with the cellular plasma membrane, and has two mainfunctions. One is the discharge of the vesicle contents into theextracellular space, and the second is the incorporation of new proteinsand lipids into the plasma membrane itself.

Exocytosis can be divided into two classes: constitutive and regulated.All eukaryotic cells exhibit constitutive exocytosis, which is marked bythe continuous fusion of the secretory vesicles after formation.Regulated exocytosis is restricted to certain cells, including exocrine,endocrine and neuronal cells. Regulated exocytosis results in theaccumulation of the secretory vesicles that fuse with the plasmamembrane only upon receipt of an appropriate signal, usually (but notalways) an increase in the cytosolic free Ca²⁺ concentration.

Regulated exocytosis is crucial to many specialized cells, and often aparticular cell can release multiple mediators from the same exocyticgranules which work in concert to produce a coordinated physiologicalresponse in the target cells. These regulated exocytic cells includeneurons (neurotransmitter release), adrenal chromaffin cells (adrenalinesecretion), pancreatic acinar cells (digestive enzyme secretion),pancreatic β-cells (insulin secretion), mast cells (histaminesecretion), mammary cells (milk protein secretion), sperm (enzymesecretion), egg cells (creation of fertilization envelope) andadipocytes (insertion of glucose transporters into the plasma membrane).In addition, current theory suggests that the basic mechanisms ofvesicle docking and fusion is conserved from yeast to the mammalianbrain.

In addition, disorders involving exocytosis are known. For example,inflammatory mediator release from mast cells leads to a variety ofdisorders, including asthma. In the United States alone, over 50 millionpeople suffer from asthma, rhinitis, or some other form of allergy.Therapy for allergy remains limited to blocking the mediators releasedby mast cells (anti-histamines), non-specific anti-inflammatory agentssuch as steroids and mast cell stabilizers which are only marginallyeffective at limiting the symtomatology of allergy. Similarly,Chediak-Higashi Syndrome (CHS) is a rare autosomal recessive disease inwhich neutrophils, monocytes and lymphocytes and most cells containgiant cytoplasmic granules. Similar disorders have been described inmice, mink, cattle, cats and killer whales, with the murine homolog ofCHS (designated beige or bg) being the best characterized. See Perou etal., J. Biol. Chem. 272(47):29790 (1997) and Barbosa et al., Nature382:262 (1996), both of which are hereby incorporated by reference.

Furthermore, it is widely believed that a wide array of psychiatricdisorders are the result of an imbalance between neurotransmitterexocytosis and mediator reuptake.

A large number of pharmaceuticals have been designed to specificallyinterfere with the exocytic mediators primarily through blockade oftheir receptors. However, this approach is limited by the fact that asingle receptor blocker cannot overcome the effects of many diversemediators.

Accordingly, it is an object of the present invention to provide methodsfor screening for alterations in exocytosis, particularly for screeningfor agents capable of mediating such exocytosis. It is also an object toprovide such screening methods wherein assay background is reduced andspecificity is increased.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides methods for screening bioactive agents for the ability to alteror modulate alterations in cellular phenotypes. The methods generallycomprise combining at least one candidate bioactive agent and apopulation of cells, sorting the cells in a FACS machine by separatingthe cells on the basis of at least three, four or five cellularparameters. The candidate agents can be part of a molecular librarycomprising fusion nucleic acids encoding the candidate bioactive agents.

In a further aspect, the present invention provides methods forscreening for alterations in exocytosis of a population of cells or insingle cells under different conditions or combined with differentbioactive agents. The methods comprise sorting the cells in a FACSmachine by assaying for alterations in at least three of the propertiesselected from the group consisting of light scattering, fluorescent dyeuptake, fluorescent dye release, annexin granule binding, surfacegranule enzyme activity, and the quantity of granule specific proteins.

Also provided herein is a method for screening for a bioactive agentcapable of modulating exocytosis in a cell. This method comprisescombining a candidate bioactive agent and a population of cells andsubjecting said cells to conditions that normally cause exocytosis. Thecells are sorted in a FACS machine by assaying for alterations in atleast three of the properties selected from the group consisting oflight scattering, fluorescent dye uptake, fluorescent dye release,annexin granule binding, surface granule enzyme activity, and thequantity of granule specific proteins. Alterations in at least one ofsaid properties as compared to cells that were not exposed to thecandidate bioactive agent indicates that said agent modulatesexocytosis.

In a preferred embodiment of the method for screening for a bioactiveagent, the properties selected include at least one property selectedfrom the group consisting of fluorescent dye release, annexin granulebinding, surface granule enzyme activity, and the quantity of granulespecific proteins.

When fluoroscent dye uptake is detected, the dye is preferably a styryldye. In the case that fluoroscent dye release is detected, the dye canbe a low pH concentration dye or a styryl dye.

In a preferred embodiment, the surface granule enzyme activity isdetected by an in situ enzymology assay or by a population based enzymeassay. The enzyme substrate can be any detectable substrate. Preferably,the enzyme substrate is coupled to a FRET construct. FRET constructsinclude two fluoroscent proteins divided by a protease site. In thiscase, the protease site is specific for a granule protease.

In a preferred embodiment, granule specific proteins are detected. Thegranule specific proteins can be any detectable protein. In oneembodiment, the granule specific proteins are fusion proteins comprisinga granule specific protein and a detectable molecule which can be a FRETconstruct.

In another preferred embodiment, a method for screening for a bioactiveagent capable of modulating exocytosis in a cell is provided whereinsaid method comprises combining at least one candidate bioactive agentand a population of cells each containing a fusion nucleic acidcomprising a nucleic acid encoding a granule-specific protein and alabel. The cells are subjected to conditions that normally causeexocytosis and the alterations in the quantity of the label is detected.Alterations in the quantity of the label indicates that the agentmodulates exocytosis. Preferably, the label is an epitope tag or afluorescent molecule. In a preferred embodiment, the fluorescentmolecule is a FRET construct.

In an additional aspect, the invention provides methods of screening forexocytosis modulators comprising combining candidate bioactive agents,cells comprising nucleic acids encoding a detectable granule-specificprotein, and an agent for detecting this protein. The cells aresubjected to conditions that normally cause exocytosis, and the presenceor absence of the protein is determined.

In another preferred embodiment, a method for screening for a bioactiveagent capable of modulating exocytosis in a cell is provided whichcomprises combining at least one candidate bioactive agent and apopulation of cells. The cells are subjected to conditions that normallycause exocytosis and a fluorescent annexin is added. Alterations in theamount of the fluorescent annexin on the surface of the cells isevaluated.

In another preferred embodiment, a method for screening for a bioactiveagent capable of modulating exocytosis in a cell is provided whichcomprises providing a population of cells wherein the cells have takenin a low pH concentration dye. The low pH concentration dye loaded cellsare combined with at least one candidate bioactive agent and subjectedto conditions that normally cause exocytosis. The release of the low pHconcentration dye is detected. Alterations in the amount of released dyeindicate that the agent modulates exocytosis.

In another preferred embodiment, a method for screening for a bioactiveagent capable of modulating exocytosis in a cell is provided whichcomprises combining at least one candidate bioactive agent and apopulation of cells. The cells are subjected to conditions that normallycause exocytosis and a fluorescent substrate specific to a granuleenzyme is added. The fluorescent substrate specific to a granule enzymeis detected, wherein alterations in the amount of the fluorescentsubstrate indicative that the agent modulates exocytosis. In a preferredembodiment, the substrate comprises a FRET construct.

In an additional aspect, the present invention provides methods andcompositions for screening for bioactive agents capable of modulatingcell cycle regulation in a cell. The method comprises combining alibrary of candidate bioactive agents and a population of cells, sortingthe cells in a FACS machine by separating the cells on the basis of atleast a cell viability assay, a proliferation assay, and a cell phaseassay.

In a further aspect, the methods comprise expressing a library of fusionnucleic acids in a library of cells. The fusion nucleic acids comprise anucleic acid encoding a candidate bioactive agent and a detectablemoiety. The cells are sorted in a FACS machine by separating the cells;when the cellular phenotype is cell cycle, the cells are sorted on thebasis of at least a cell viability assay, an expression assay, aproliferation assay, and a cell phase assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C schematically depict three retroviral constructionsof Example 1. FIG. 1A includes the CRU5-GFP-p21 construction, comprisinga CRU5 promoter, the ψ-retroviral packaging signal, the coding regionfor GFP, fused to the coding region of p21, followed by an LTR. FIG. 1Bdepicts the CRU5-GFP-p21C construction, which includes the C-terminal 24amino acids of p21. FIG. 1C depicts the CRU5-GFP-pUCmut construct, whichis a mutant version of CRU5-p21C with 3 alanine substitutions.

FIGS. 2A, 2B, 2C and 2D depict the results of the experiments ofExample 1. FIG. 2A depicts a viability assay utilizing forward and sidescatter. Cells exhibiting a characteristic ratio are collected. FIG. 2Bshows the fluorescence of the GFP of the vectors. FIG. 2C depicts theuse of PKH26, an inclusion dye, in a proliferation assay; the cellscontaining p21, a protein known to arrest cells, remain brightlyfluorescent, while the control cells continue to proliferate, thusdiluting the dye and losing fluorescence. FIG. 2D depicts the use ofHoechst 33342 in a cell phase assay.

FIG. 3 depicts the effect of AraC treatment on Jurkat cells infectedwith p21, an agent that arrests cells in the G1 phase. AraC is anucleotide analog that is toxic to dividing cells. Thus, those cellsthat are cell cycle arrested survive. The lower line depicts Jurkatcells without the p21 insert, and the upper line depicts Jurkat cellswith the p21 insert.

FIGS. 4A and 4B depict bar graphs showing the results of a populationbased exocytic enzyme activity assay for exocytosis. FIG. 4A showsglucuronidase or hexosaminidase activity in the supernatant of cellscombined with DMSO (−) or ionomycin (+). FIG. 4B shows hexosaminidaseactivity in the supernatant of cells sensitized with varying amounts ofIgE anti-DNP and stimulated with increasing amounts of the antigenBSA-DNP.

FIGS. 5A-5F show exocytic light scatter changes observed on the flowcytometer, side scatter vs. forward scatter, plotted as bivariatehistograms for RBL-2H3 cells (FIGS. 5A and 5D) and MC-9 cells (FIGS. 5B,5C, 5E and 5F). After stimulation with an ionophore, the cells wereobserved at 0 minutes (FIGS. 5A and 5C), 5 minutes (FIG. 5E), 10 minutes(FIG. 5D), and 30 minutes (FIGS. 5B and 5F).

FIGS. 6A-6E show graphs of the results of a styryl dye assay to detectexocytosis by FACS. Cells were combined with (left peaks) DMSO or (rightpeaks) ionomycin in the presence of either FM 4-64 (FIGS. 6A and 6B) orFM 1-43 (FIGS. 6C, 6D and 6E). FIGS. 6A and 6C show cells detected influorescence channel 1. FIGS. 6B and 6D show cells detected influorescence channel 3. FIG. 6E shows the mean channel shift detected inthe flow cytometer in fluorescence channel 1 plotted as a bar graphwherein cells were preincubated with varying doses of the PI-3 kinaseinhibitor wortmannin prior to administration of an ionophore (bars 1-4)or DMSO (bar 5) in the presence of FM 1-43.

FIGS. 7A-7D show graphs depicting the results of an annexin-V detectionassay of exocytosis by FACS. Cells were combined with either DMSO (FIGS.7A and 7B) or ionomycin (FIGS. 7C and 7D) and then stained with bothpropidium iodide (FIGS. 7A and 7C) and annexin-V-FITC (FIGS. 7B and 7D).

FIGS. 8A-8C show graphs indicating the results of an in situ enzymologyassay of exocytosing cells visualized by FACS. Cells were combined withDMSO (FIG. 8A) or an ionophore (FIGS. 8B and 8C) and then stained for insitu glucuronidase activity. FIG. 8C shows the pH profile of the cellsurface enzymatic activity wherein the bar graphs represent thepercentage of maximal signal, as measured by mean channel shift in theflow cytometer, observed.

FIG. 9 is a histogram of fluorescence intensity detected in channel 1showing cells loaded with LYSOTRACKER GREEN™, combined with either DMSO(left) or ionomycin (right) and viewed in the flow cytometer.

FIGS. 10A-10H show the results of a multiparameter analysis includingdetection of LYSOTRACKER GREEN™, annexin-V-APC and forward and sidescatter. FIGS. 10A-10D and 10E-10H each show cells treated withincreasing doses of ionomycin and observed in the flow cytometer withfour parameters simultaneously. The cells were loaded with low pHconcentration dye, stimulated and stained with annexin-V-APC. FIGS.10A-10D show bivariate histograms of side vs. forward light scatter andFIGS. 10E-10H show bivariate histograms of annexin-V-APC vs. low pHconcentration dye signals.

FIG. 11 shows a graph of cells stimulated in the presence of FM 1-43 andannexin-V-APC stained. At various timepoints after ionomycin stimulationthe cells were analyzed by flow cytometry and the supernatant forenzymatic activity (cell supernatant). The parameters forward scatter,FM-143, annexin-V-APC, and hexosaminidase are plotted on the graphrelative to the maximal response for each parameter. For calciumsignaling, a separate tube of cells was loaded with Fluo-3 and underwentthe identical procedure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the detection of alterations incellular phenotypes, such as cell cycle regulation, exocytosis, smallmolecule toxicity, cell surface receptor expression, enzyme expression,etc. by evaluating or assaying a variety of cellular parameters,generally through the use of a fluorescence-activated cell sorter (FACS)machine. There are a number of parameters that can be measured to allowdetection of alterations in a variety of cellular phenotypes as is morefully outlined below. By assaying a plurality of these parameters eithersequentially or preferably simultaneously, rapid and accurate screeningmay be done.

In a preferred embodiment, the methods outlined herein are used toscreen for modulators of cellular phenotypes. Cellular phenotypes thatmay be assayed include, but are not limited to, cellular apoptosis,including cell cycle regulation, exocytosis, toxicity to smallmolecules, the expression of any number of moieties including receptors(particularly cell surface receptors), adhesion molecules, cytokinesecretion, protein-protein interactions, etc.

In a preferred embodiment, the methods are used to evaluate cell cycleregulation. In this embodiment, preferred cellular parameters or assaysare cell viability assays, assays to determine whether cells arearrested at a particular cell cycle stage (“cell proliferation assays”),and assays to determine at which cell stage the cells have arrested(“cell phase assays”). By assaying or measuring one or more of theseparameters, it is possible to detect not only alterations in cell cycleregulation, but alterations of different steps of the cell cycleregulation pathway. This may be done to evaluate native cells, forexample to quantify the aggressiveness of a tumor cell type, or toevaluate the effect of candidate drug agents that are being tested fortheir effect on cell cycle regulation. In this manner, rapid, accuratescreening of candidate agents may be performed to identify agents thatmodulate cell cycle regulation.

Thus, the present methods are useful to elucidate bioactive agents thatcan cause a population of cells to either move out of one growth phaseand into another, or arrest in a growth phase. In some embodiments, thecells are arrested in a particular growth phase, and it is desirable toeither get them out of that phase or into a new phase. Alternatively, itmay be desirable to force a cell to arrest in a phase, for example G1,rather than continue to move through the cell cycle. Similarly, it maybe desirable in some circumstances to accelerate a non-arrested butslowly moving population of cells into either the next phase or justthrough the cell cycle, or to delay the onset of the next phase. Forexample, it may be possible to alter the activities of certain enzymes,for example kinases, phosphatases, proteases or ubiquitination enzymes,that contribute to initiating cell phase changes.

In a preferred embodiment, the methods outlined herein are done on cellsthat are not arrested in the G1 phase; that is, they are rapidly oruncontrollably growing and replicating, such as tumor cells. In thismanner, candidate agents are evaluated to find agents that can alter thecell cycle regulation, i.e. cause the cells to arrest at cell cyclecheckpoints, such as in G1 (although arresting in other phases such asS, G2 or M are also desirable). Alternatively, candidate agents areevaluated to find agents that can cause proliferation of a population ofcells, i.e. that allow cells that are generally arrested in G1 to startproliferating again; for example, peripheral blood cells, terminallydifferentiated cells, stem cells in culture, etc.

Accordingly, in a preferred embodiment, the invention provides methodsfor screening for alterations in cell cycle regulation of a populationof cells. “Alteration” and “modulation” (used herein interchangeably),as used herein can include both increases and decreases in the parameteror phenotype being measured. By “alteration” or “modulation” in thecontext of cell cycle regulation, is generally meant one of two things.In a preferred embodiment, the alteration results in a change in thecell cycle of a cell, i.e. a proliferating cell arrests in any one ofthe phases, or an arrested cell moves out of its arrested phase andstarts the cell cycle, as compared to another cell or in the same cellunder different conditions. Alternatively, the progress of a cellthrough any particular phase may be altered; that is, there may be anacceleration or delay in the length of time it takes for the cells tomove thorough a particular growth phase. For example, the cell may benormally undergo a G1 phase of several hours; the addition of an agentmay prolong the G1 phase.

The measurements can be determined wherein all of the conditions are thesame for each measurement, or under various conditions, with or withoutbioactive agents, or at different stages of the cell cycle process. Forexample, a measurement of cell cycle regulation can be determined in acell population wherein a candidate bioactive agent is present andwherein the candidate bioactive agent is absent. In another example, themeasurements of cell cycle regulation are determined wherein thecondition or environment of the populations of cells differ from oneanother. For example, the cells may be evaluated in the presence orabsence of physiological signals, for example hormones, antibodies,peptides, antigens, cytokines, growth factors, action potentials,pharmacological agents (i.e. chemotherapeutics, etc.), or other cells(i.e. cell-cell contacts). In another example, the measurements of cellcycle regulation are determined at different stages of the cell cycleprocess. In yet another example, the measurements of cell cycleregulation are taken wherein the conditions are the same, and thealterations are between one cell or cell population and another cell orcell population.

By a “population of cells” or “library of cells” or “plurality of cells”herein is meant at least two cells, with at least about 10³ beingpreferred, at least about 10⁶ being particularly preferred, and at leastabout 10⁸ to 10⁹ being especially preferred. The population or samplecan contain a mixture of different cell types from either primary orsecondary cultures although samples containing only a single cell typeare preferred, for example, the sample can be from a cell line,particularly tumor cell lines (particularly when, as outlined below. Thecells may be in any cell phase, either synchronously or not, includingM, G1, S, and G2. In a preferred embodiment, cells that are replicatingor proliferating are used; this may allow the use of retroviral vectorsfor the introduction of candidate bioactive agents. Alternatively,non-replicating cells may be used, and other vectors (such as adenovirusand lentivirus vectors) can be used. In addition, although not required,the cells are compatible with dyes and antibodies.

Preferred cell types for use in the invention will vary with thecellular phenotype to be modulated. Suitable cells include, but are notlimited to, mammalian cells, including animal (rodents, including mice,rats, hamsters and gerbils), primates, and human cells, particularlyincluding tumor cells of all types, including breast, skin, lung,cervix, colonrectal, leukemia, brain, etc. As outlined below, additionalcell types may be used for screening for exocytosis.

In a preferred embodiment, the cell cycle regulation methods comprisesorting the cells in a FACS machine by assaying several different cellparameters, including, but not limited to, cell viability, cellproliferation, and cell phase.

In a preferred embodiment, cell viability is assayed, to ensure that alack of cellular change is due to experimental conditions (i.e. theintroduction of a candidate bioactive agent) not cell death. There are avariety of suitable cell viability assays which can be used, including,but not limited to, light scattering, viability dye staining, andexclusion dye staining.

In a preferred embodiment, a light scattering assay is used as theviability assay, as is well known in the art. When viewed in the FACS,cells have particular characteristics as measured by their forward and90 degree (side) light scatter properties. These scatter propertiesrepresent the size, shape and granule content of the cells. Theseproperties account for two parameters to be measured as a readout forthe viability. Briefly, the DNA of dying or dead cells generallycondenses, which alters the 90° scatter; similarly, membrane blebbingcan alter the forward scatter. Alterations in the intensity of lightscattering, or the cell-refractive index indicate alterations inviability.

Thus, in general, for light scattering assays, a live cell population ofa particular cell type is evaluated to determine it's forward and sidescattering properties. This sets a standard for scattering that cansubsequently be used.

In a preferred embodiment, the viability assay utilizes a viability dye.There are a number of known viability dyes that stain dead or dyingcells, but do not stain growing cells. For example, annexin V is amember of a protein family which displays specific binding tophospholipid (phosphotidylserine) in a divalent ion dependent manner.This protein has been widely used for the measurement of apoptosis(programmed cell death) as cell surface exposure of phosphatidylserineis a hallmark early signal of this process. Suitable viability dyesinclude, but are not limited to, annexin, ethidium homodimer-1, DEADRed, propidium iodide, SYTOX Green, etc., and others known in the art;see the Molecular Probes Handbook of Fluorescent Probes and ResearchChemicals, Haugland, Sixth Edition, hereby incorporated by reference;see Apoptosis Assay on page 285 in particular, and Chapter 16.

Protocols for viability dye staining for cell viability are known, seeMolecular Probes catalog, supra. In this embodiment, the viability dyesuch as annexin is labeled, either directly or indirectly, and combinedwith a cell population. Annexin is commercially available, i.e., fromPharMingen, San Diego, Calif., or Caltag Laboratories, Millbrae, Calif.Preferably, the viability dye is provided in a solution wherein the dyeis in a concentration of about 100 ng/ml to about 500 ng/ml, morepreferably, about 500 ng/ml to about 1 μg/ml, and most preferably, fromabout 1 μg/ml to about 5 μg/ml. In a preferred embodiment, the viabilitydye is directly labeled; for example, annexin may be labeled with afluorochrome such as fluorecein isothiocyanate (FITC), Alexa dyes,TRITC, AMCA, APC, tri-color, Cy-5, and others known in the art orcommercially available. In an alternate preferred embodiment, theviability dye is labeled with a first label, such as a hapten such asbiotin, and a secondary fluorescent label is used, such as fluorescentstreptavidin. Other first and second labeling pairs can be used as willbe appreciated by those in the art.

Once added, the viability dye is allowed to incubate with the cells fora period of time, and washed, if necessary. The cells are then sorted asoutlined below to remove the non-viable cells.

In a preferred embodiment, exclusion dye staining is used as theviability assay. Exclusion dyes are those which are excluded from livingcells, i.e. they are not taken up passively (they do not permeate thecell membrane of a live cell). However, due to the permeability of deador dying cells, they are taken up by dead cells. Generally, but notalways, the exclusion dyes bind to DNA, for example via intercalation.Preferably, the exclusion dye does not fluoresce, or fluoresces poorly,in the absence of DNA; this eliminates the need for a wash step.Alternatively, exclusion dyes that require the use of a secondary labelmay also be used. Preferred exclusion dyes include, but are not limitedto, ethidium bromide; ethidium homodimer-1; propidium iodine; SYTOXgreen nucleic acid stain; Calcein AM, BCECF AM; fluorescein diacetate;TOTO® and TO-PRO™ (from Molecular Probes; supra, see chapter 16) andothers known in the art.

Protocols for exclusion dye staining for cell viability are known, seethe Molecular Probes catalog, supra. In general, the exclusion dye isadded to the cells at a concentration of from about 100 ng/ml to about500 ng/ml, more preferably, about 500 ng/ml to about 1 μg/ml, and mostpreferably, from about 0.1 μg/ml to about 5 μg/ml, with about 0.5 μg/mlbeing particularly preferred. The cells and the exclusion dye areincubated for some period of time, washed, if necessary, and then thecells sorted as outlined below, to remove non-viable cells from thepopulation.

In addition, there are other cell viability assays which may be run,including for example enzymatic assays, which can measure extracellularenzymatic activity of either live cells (i.e. secreted proteases, etc.),or dead cells (i.e. the presence of intracellular enzymes in the media;for example, intracellular proteases, mitochondrial enzymes, etc.). Seethe Molecular Probes Handbook of Fluorescent Probes and ResearchChemicals, Haugland, Sixth Edition, hereby incorporated by reference;see chapter 16 in particular.

In a preferred embodiment, at least one cell viability assay is run,with at least two different cell viability assays being preferred, whenthe fluors are compatible. When only 1 viability assay is run, apreferred embodiment utilizes light scattering assays (both forward andside scattering). When two viability assays are run, preferredembodiments utilize light scattering and dye exclusion, with lightscattering and viability dye staining also possible, and all three beingdone in some cases as well. Viability assays thus allow the separationof viable cells from non-viable or dying cells.

In addition to a cell viability assay, a preferred embodiment utilizes acell proliferation assay. By “proliferation assay” herein is meant anassay that allows the determination that a cell population is eitherproliferating, i.e. replicating, or not replicating.

In a preferred embodiment, the proliferation assay is a dye inclusionassay. A dye inclusion assay relies on dilution effects to distinguishbetween cell phases. Briefly, a dye (generally a fluorescent dye asoutlined below) is introduced to cells and taken up by the cells. Oncetaken up, the dye is trapped in the cell, and does not diffuse out. Asthe cell population divides, the dye is proportionally diluted. That is,after the introduction of the inclusion dye, the cells are allowed toincubate for some period of time; cells that lose fluorescence over timeare dividing, and the cells that remain fluorescent are arrested in anon-growth phase.

Generally, the introduction of the inclusion dye may be done in one oftwo ways. Either the dye cannot passively enter the cells (e.g. it ischarged), and the cells must be treated to take up the dye; for examplethrough the use of a electric pulse. Alternatively, the dye canpassively enter the cells, but once taken up, it is modified such thatit cannot diffuse out of the cells. For example, enzymatic modificationof the inclusion dye may render it charged, and thus unable to diffuseout of the cells. For example, the Molecular Probes CellTracker™ dyesare fluorescent chloromethyl derivatives that freely diffuse into cells,and then glutathione S-transferase-mediated reaction produces membraneimpermeant dyes.

Suitable inclusion dyes include, but are not limited to, the MolecularProbes line of CellTracker™ dyes, including, but not limited toCellTracker™ Blue, CellTracker™ Yellow-Green, CellTracker™ Green,CellTracker™ Orange, PKH26 (Sigma), and others known in the art; see theMolecular Probes Handbook, supra; chapter 15 in particular.

In general, inclusion dyes are provided to the cells at a concentrationranging from about 100 ng/ml to about 5 μg/ml, with from about 500 ng/mlto about 1 μg/ml being preferred. A wash step may or may not be used. Ina preferred embodiment, a candidate bioactive agent is combined with thecells as described herein. The cells and the inclusion dye are incubatedfor some period of time, to allow cell division and thus dye dilution.The length of time will depend on the cell cycle time for the particularcells; in general, at least about 2 cell divisions are preferred, withat least about 3 being particularly preferred and at least about 4 beingespecially preferred. The cells are then sorted as outlined below, tocreate populations of cells that are replicating and those that are not.As will be appreciated by those in the art, in some cases, for examplewhen screening for anti-proliferation agents, the bright (i.e.fluorescent) cells are collected; in other embodiments, for example forscreening for proliferation agents, the low fluorescence cells arecollected. Alterations are determined by measuring the fluorescence ateither different time points or in different cell populations, andcomparing the determinations to one another or to standards.

In a preferred embodiment, the proliferation assay is an antimetaboliteassay. In general, antimetabolite assays find the most use when agentsthat cause cellular arrest in G1 or G2 resting phase is desired. In anantimetabolite proliferation assay, the use of a toxic antimetabolitethat will kill dividing cells will result in survival of only thosecells that are not dividing. Suitable antimetabolites include, but arenot limited to, standard chemotherapeutic agents such as methotrexate,cisplatin, taxol, hydroxyurea, nucleotide analogs such as AraC, etc. Inaddition, antimetabolite assays may include the use of genes that causecell death upon expression.

The concentration at which the antimetabolite is added will depend onthe toxicity of the particular antimetabolite, and will be determined asis known in the art. The antimetabolite is added and the cells aregenerally incubated for some period of time; again, the exact period oftime will depend on the characteristics and identity of theantimetabolite as well as the cell cycle time of the particular cellpopulation. Generally, a time sufficient for at least one cell divisionto occur.

In a preferred embodiment, at least one proliferation assay is run, withmore than one being preferred. Thus, a proliferation assay results in apopulation of proliferating cells and a population of arrested cells.

In a preferred embodiment, either after or simultaneously with one ormore of the proliferation assays outlined above, at least one cell phaseassay is done. A “cell phase” assay determines at which cell phase thecells are arrested, M, G1, S, or G2.

In a preferred embodiment, the cell phase assay is a DNA binding dyeassay. Briefly, a DNA binding dye is introduced to the cells, and takenup passively. Once inside the cell, the DNA binding dye binds to DNA,generally by intercalation, although in some cases, the dyes can beeither major or minor groove binding compounds. The amount of dye isthus directly correlated to the amount of DNA in the cell, which variesby cell phase; G2 and M phase cells have twice the DNA content of G1phase cells, and S phase cells have an intermediate amount, depending onat what point in S phase the cells are. Suitable DNA binding dyes arepermeant, and include, but are not limited to, Hoechst 33342 and 33258,acridine orange, 7-AAD, LDS 751, DAPI, and SYTO 16, Molecular ProbesHandbook, supra; chapters 8 and 16 in particular.

In general, the DNA binding dyes are added in concentrations rangingfrom about 1 μg/ml to about 5 μg/ml. The dyes are added to the cells andallowed to incubate for some period of time; the length of time willdepend in part on the dye chosen. In one embodiment, measurements aretaken immediately after addition of the dye. The cells are then sortedas outlined below, to create populations of cells that contain differentamounts of dye, and thus different amounts of DNA; in this way, cellsthat are replicating are separated from those that are not. As will beappreciated by those in the art, in some cases, for example whenscreening for anti-proliferation agents, cells with the leastfluorescence (and thus a single copy of the genome) can be separatedfrom those that are replicating and thus contain more than a singlegenome of DNA. Alterations are determined by measuring the fluorescenceat either different time points or in different cell populations, andcomparing the determinations to one another or to standards.

In a preferred embodiment, the cell phase assay is a cyclin destructionassay. In this embodiment, prior to screening (and generally prior tothe introduction of a candidate bioactive agent, as outlined below), afusion nucleic acid is introduced to the cells. The fusion nucleic acidcomprises nucleic acid encoding a cyclin destruction box and a nucleicacid encoding a detectable molecule. “Cyclin destruction boxes” areknown in the art and are sequences that cause destruction via theubiquitination pathway of proteins containing the boxes duringparticular cell phases. That is, for example, G1 cyclins may be stableduring G1 phase but degraded during S phase due to the presence of a G1cyclin destruction box. Thus, by linking a cyclin destruction box to adetectable molecule, for example green fluorescent protein, the presenceor absence of the detectable molecule can serve to identify the cellphase of the cell population. In a preferred embodiment, multiple boxesare used, preferably each with a different fluor, such that detection ofthe cell phase can occur.

A number of cyclin destruction boxes are known in the art, for example,cyclin A has a destruction box comprising the sequence RTVLGVIGD; thedestruction box of cyclin B1 comprises the sequence RTALGDIG. SeeGlotzer et al., Nature 349:132-138 (1991). Other destruction boxes areknown as well: YMTVSIIDRFMQDSCVPKKMLQLVGVT (rat cyclin B);KFRLLQETMYMTVSIIDRFMQNSCVPKK (mouse cyclin B);RAILIDWLIQVQMKFRLLQETMYMTVS (mouse cyclin B1);DRFLQAQLVCRKKLQWGITALLLASK (mouse cyclin B2); and MSVLRGKLQLVGTAAMLL(mouse cyclin A2).

The nucleic acid encoding the cyclin destruction box is operably linkedto nucleic acid encoding a detectable molecule. The fusion proteins areconstructed by methods known in the art. For example, the nucleic acidsencoding the destruction box is ligated to a nucleic acid encoding adetectable molecule. By “detectable molecule” herein is meant a moleculethat allows a cell or compound comprising the detectable molecule to bedistinguished from one that does not contain it, i.e., an epitope,sometimes called an antigen TAG, a specific enzyme, or a fluorescentmolecule. Preferred fluorescent molecules include but are not limited togreen fluorescent protein (GFP), blue fluorescent protein (BFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), and enzymesincluding luciferase and β-galactosidase. When antigen TAGs are used,preferred embodiments utilize cell surface antigens. The epitope ispreferably any detectable peptide which is not generally found on thecytoplasmic membrane, although in some instances, if the epitope is onenormally found on the cells, increases may be detected, although this isgenerally not preferred. Similarly, enzymatic detectable molecules mayalso be used; for example, an enzyme that generates a novel orchromogenic product.

Accordingly, the results of sorting after cell phase assays generallyresult in at least two populations of cells that are in different cellphases.

In a preferred embodiment, the methods are used to screen candidatebioactive agents for the ability to modulate cell cycle regulation,including the activation or suppression of cell cycle checkpointpathways and ameliorating checkpoint defects. The candidate bioactiveagent can be added to the cell population exogenously or can beintroduced into the cells as described further herein.

The term “candidate bioactive agent” or “exogeneous compound” as usedherein describes any molecule, e.g., protein, small organic molecule,carbohydrates (including polysaccharides), polynucleotide, lipids, etc.Generally a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration or below the level ofdetection. In addition, positive controls can be used. For example, inthe cell cycling assays, agents known to alter cell cycling may be used.For example, p21 is a molecule known to arrest cells in the G1 cellphase, by binding G1 cyclin-CDK complexes. Similarly, for exocytosis,compounds known to induce exocytosis can be used as is more fullyoutlined below.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty adds, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In a preferred embodiment, the candidate bioactive agents are proteins.By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations. Chemical blocking groups orother chemical substituents may also be added.

In a preferred embodiment, the candidate bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eukaryotic proteins may be madefor screening in the systems described herein. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In a preferred embodiment, the candidate bioactive agents are peptidesof from about 5 to about 30 amino acids, with from about 5 to about 20amino acids being preferred, and from about 7 to about 15 beingparticularly preferred. The peptides may be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. By “randomized” or grammatical equivalents herein ismeant that each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. Since generally these randompeptides (or nucleic acids, discussed below) are chemically synthesized,they may incorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteins ornucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the candidate bioactive agents are nucleicacids. By “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein means at least two nucleotides covalently linked together. Anucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925(1993) and references therein; Letsinger, J. Org. Chem., 35:3800 (1970);Sprinzl, et al. Eur. J. Biochem., 81:579 (1977); Letsinger, et al.,Nucl. Acids Res., 14:3487 (1986); Sawai, et al., Chem. Lett., 805(1984), Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); andPauwels, et al., Chemica Scripta, 26:141 (1986)), phosphorothioate (Mag,et al., Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No.5,644,048), phosphorodithioate (Briu, et al., J. Am. Chem. Soc.,111:2321 (1989)), O-methylphosphoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc., 114:1895 (1992); Meier, et al. Chem. Int. Ed. Engl.,31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson, et al.,Nature, 380:207 (1996), all of which are incorporated by reference)).Other analog nucleic acids include those with positive backbones(Denpcy, et al., Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionicbackbones (U.S. Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141;and 4,469,863; Kiedrowshi, at al., Angew. Chem. Intl. Ed, English,30:423 (1991); Letsinger, et al., J. Am. Chem. Soc. 110:4470 (1988);Letsinger, et al., Nucleoside & Nucleotide, 13:1597 (1994); Chapters 2and 3, ASC Symposium Series 580, “Carbohydrate Modifications inAntisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, etal., Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,at al., Chem. Soc. Rev., (1995) pp. 169-176). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Allof these references are hereby expressly incorporated by reference.These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of additional moieties such as labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments. In addition, mixtures of naturally occurring nucleic acidsand analogs can be made. Alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. The nucleic acids may be single stranded or doublestranded, as specified, or contain portions of both double stranded orsingle stranded sequence. The nucleic acid may be DNA, both genomic andcDNA, RNA or a hybrid, where the nucleic acid contains any combinationof deoxyribo- and ribo-nucleotides, and any combination of bases,including uracil, adenine, thymine, cytosine, guanine, inosine,xathanine hypoxathanine, isocytosine, isoguanine, etc.

As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eukaryotic genomes may be used as is outlined above forproteins.

In a preferred embodiment, the candidate bioactive agents are organicchemical moieties, a wide variety of which are available in theliterature.

In a preferred embodiment, a library of different candidate bioactiveagents are used. Preferably, the library should provide a sufficientlystructurally diverse population of randomized agents to effect aprobabilistically sufficient range of diversity to allow binding to aparticular target. Accordingly, an interaction library should be largeenough so that at least one of its members will have a structure thatgives it affinity for the target. Although it is difficult to gauge therequired absolute size of an interaction library, nature provides a hintwith the immune response: a diversity of 10⁷-10⁸ different antibodiesprovides at least one combination with sufficient affinity to interactwith most potential antigens faced by an organism. Published in vitroselection techniques have also shown that a library size of 10⁷ to 10⁸is sufficient to find structures with affinity for the target. A libraryof all combinations of a peptide 7 to 20 amino acids in length, such asgenerally proposed herein, has the potential to code for 20⁷ (10⁹) to20²⁰. Thus, with libraries of 10′ to 10⁸ different molecules the presentmethods allow a “working” subset of a theoretically complete interactionlibrary for 7 amino acids, and a subset of shapes for the 20²⁰ library.Thus, in a preferred embodiment, at least 10⁶, preferably at least 10′,more preferably at least 10⁸ and most preferably at least 10⁸ differentsequences are simultaneously analyzed in the subject methods. Preferredmethods maximize library size and diversity.

The candidate bioactive agents are combined or added to a cell orpopulation of cells. Suitable cell types for different embodiments areoutlined above. The candidate bioactive agent and the cells arecombined. As will be appreciated by those in the art, this mayaccomplished in any number of ways, including adding the candidateagents to the surface of the cells, to the media containing the cells,or to a surface on which the cells are growing or in contact with;adding the agents into the cells, for example by using vectors that willintroduce the agents into the cells (i.e. when the agents are nucleicacids or proteins).

In a preferred embodiment, the candidate bioactive agents are eithernucleic acids or proteins (proteins in this context includes proteins,oligopeptides, and peptides) that are introduced into the host cellsusing vectors, including viral vectors. The choice of the vector,preferably a viral vector, will depend on the cell type. When the cellsare replicating, retroviral vectors are used as is more fully describedbelow. When the cells are not replicating (i.e. they are arrested in oneof the growth phases), other viral vectors may be used, includinglentiviral and adenoviral vectors.

In a preferred embodiment, the cells are either replicating or can beinduced to replicate, and retroviral vectors are used to introducecandidate bioactive agents to the cells, as is generally outlined in PCTUS97/01019 and PCT US97/01048, both of which are expressly incorporatedby reference. Generally, a library of retroviral vectors is made usingretroviral packaging cell lines that are helper-defective and arecapable of producing all the necessary trans proteins, including gag,pol and env, and RNA molecules that have in cis the ψ packaging signal.Briefly, the library is generated in a retrovirus DNA constructbackbone; standard oligonucleotide synthesis is done to generate eitherthe candidate agent or nucleic acid encoding a protein, for example arandom peptide, using techniques well known in the art. After generationof the DNA library, the library is cloned into a first primer. The firstprimer serves as a “cassette”, which is inserted into the retroviralconstruct. The first primer generally contains a number of elements,including for example, the required regulatory sequences (e.g.translation, transcription, promoters, etc), fusion partners,restriction endonuclease (cloning and subcloning) sites, stop codons(preferably in all three frames), regions of complementarity for secondstrand priming (preferably at the end of the stop codon region as minordeletions or insertions may occur in the random region), etc.

A second primer is then added, which generally consists of some or allof the complementarity region to prime the first primer and optionalnecessary sequences for a second unique restriction site for subcloning.DNA polymerase is added to make double-stranded oligonucleotides. Thedouble-stranded oligonucleotides are cleaved with the appropriatesubcloning restriction endonucleases and subcloned into the targetretroviral vectors, described below.

Any number of suitable retroviral vectors may be used. Generally, theretroviral vectors may include: selectable marker genes as is more fullydescribed below; promoters driving expression of a second gene, placedin sense or anti-sense relative to the 5′ LTR; CRU5 (a synthetic LTR),tetracycline regulation elements in SIN, cell specific promoters, etc.

Preferred retroviral vectors include a vector based on the murine stemcell virus (MSCV) (see Hawley et al., Gene Therapy 1:136 (1994)) and amodified MFG virus (Rivere et al., Genetics 92:6733 (1995)), and pBABE,outlined in PCT US97/01019.

The retroviruses may include inducible and constitutive promoters forthe expression of the candidate agent. For example, there are situationswherein it is necessary to induce peptide expression only during certainphases of the selection process, or only in certain cell phases (i.e.using phase specific promoters, such as E2F responsive promoter, p53responsive promoter, cyclin promoters, etc.). A large number of bothinducible and constitutive promoters are known.

In addition, it is possible to configure a retroviral vector to allowinducible expression of retroviral inserts after integration of a singlevector in target cells; importantly, the entire system is containedwithin the single retrovirus. Tet-inducible retroviruses have beendesigned incorporating the Self-Inactivating (SIN) feature of 3′ LTRenhancer/promoter retroviral deletion mutant (Hoffman et al., PNAS USA93:5185 (1996)). Expression of this vector in cells is virtuallyundetectable in the presence of tetracycline or other active analogs.However, in the absence of Tet, expression is turned on to maximumwithin 48 hours after induction, with uniform increased expression ofthe whole population of cells that harbor the inducible retrovirus,indicating that expression is regulated uniformly within the infectedcell population. A similar, related system uses a mutated TetDNA-binding domain such that it bound DNA in the presence of Tet, andwas removed in the absence of Tet. Either of these systems is suitable.

In a preferred embodiment, the candidate bioactive agents are linked toa fusion partner. By “fusion partner” or “functional group” herein ismeant a sequence that is associated with the candidate bioactive agent,that confers upon all members of the library in that class a commonfunction or ability. Fusion partners can be heterologous (i.e. notnative to the host cell), or synthetic (not native to any cell).Suitable fusion partners include, but are not limited to: a)presentation structures, as defined below, which provide the candidatebioactive agents in a conformationally restricted or stable form; b)targeting sequences, defined below, which allow the localization of thecandidate bioactive agent into a subcellular or extracellularcompartment; c) rescue sequences as defined below, which allow thepurification or isolation of either the candidate bioactive agents orthe nucleic acids encoding them; d) stability sequences, which conferstability or protection from degradation to the candidate bioactiveagent or the nucleic acid encoding it, for example resistance toproteolytic degradation; e) dimerization sequences, to allow for peptidedimerization; or f) any combination of a), b), c), d), and e), as wellas linker sequences as needed.

In a preferred embodiment, the fusion partner is a presentationstructure. By “presentation structure” or grammatical equivalents hereinis meant a sequence, which, when fused to candidate bioactive agents,causes the candidate agents to assume a conformationally restrictedform. Proteins interact with each other largely through conformationallyconstrained domains. Although small peptides with freely rotating aminoand carboxyl termini can have potent functions as is known in the art,the conversion of such peptide structures into pharmacologic agents isdifficult due to the inability to predict side-chain positions forpeptidomimetic synthesis. Therefore the presentation of peptides inconformationally constrained structures will benefit both the latergeneration of pharmaceuticals and will also likely lead to higheraffinity interactions of the peptide with the target protein. This facthas been recognized in the combinatorial library generation systemsusing biologically generated short peptides in bacterial phage systems.A number of workers have constructed small domain molecules in which onemight present randomized peptide structures.

While the candidate bioactive agents may be either nucleic acid orpeptides, presentation structures are preferably used with peptidecandidate agents. Thus, synthetic-presentation structures, i.e.artificial polypeptides, are capable of presenting a randomized peptideas a conformationally-restricted domain. Generally such presentationstructures comprise a first portion joined to the N-terminal end of therandomized peptide, and a second portion joined to the C-terminal end ofthe peptide; that is, the peptide is inserted into the presentationstructure, although variations may be made, as outlined below. Toincrease the functional isolation of the randomized expression product,the presentation structures are selected or designed to have minimalbiologically activity when expressed in the target cell.

Preferred presentation structures maximize accessibility to the peptideby presenting it on an exterior loop. Accordingly, suitable presentationstructures include, but are not limited to, minibody structures, loopson beta-sheet turns and coiled-coil stem structures in which residuesnot critical to structure are randomized, zinc-finger domains,cysteine-linked (disulfide) structures, transglutaminase linkedstructures, cyclic peptides, B-loop structures, helical barrels orbundles, leucine zipper motifs, etc.

In a preferred embodiment, the presentation structure is a coiled-coilstructure, allowing the presentation of the randomized peptide on anexterior loop. See, for example, Myszka et al., Biochem. 33:2362-2373(1994), hereby incorporated by reference). Using this systeminvestigators have isolated peptides capable of high affinityinteraction with the appropriate target. In general, coiled-coilstructures allow for between 6 to 20 randomized positions.

A preferred coiled-coil presentation structure is as follows:MGCAALESEVSALESEVASLESEVAALGRGDMPLAAVKSKLSAVKSKLASVKSKLAACGPP. Theunderlined regions represent a coiled-coil leucine zipper region definedpreviously (see Martin et al., EMBO J. 13(22):5303-5309 (1994),incorporated by reference). The bolded GRGDMP region represents the loopstructure and when appropriately replaced with randomized peptides (i.e.candidate bioactive agents, generally depicted herein as (X)_(n), whereX is an amino acid residue and n is an integer of at least 5 or 6) canbe of variable length. The replacement of the bolded region isfacilitated by encoding restriction endonuclease sites in the underlinedregions, which allows the direct incorporation of randomizedoligonucleotides at these positions. For example, a preferred embodimentgenerates a XhoI site at the double underlined LE site and a Hind IIIsite at the double-underlined KL site.

In a preferred embodiment, the presentation structure is a minibodystructure. A “minibody” is essentially composed of a minimal antibodycomplementarity region. The minibody presentation structure generallyprovides two randomizing regions that in the folded protein arepresented along a single face of the tertiary structure. See for exampleBianchi et al., J. Mol. Biol. 236(2):649-59 (1994), and references citedtherein, all of which are incorporated by reference). Investigators haveshown this minimal domain is stable in solution and have used phageselection systems in combinatorial libraries to select minibodies withpeptide regions exhibiting high affinity, Kd=10⁻⁷, for thepro-inflammatory cytokine IL-6.

A preferred minibody presentation structure is as follows:MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRYIVSRDTSQSILYLQKKKG PP.The bold, underline regions are the regions which may be randomized. Theitalized phenylalanine must be invariant in the first randomizingregion. The entire peptide is cloned in a three-oligonucleotidevariation of the coiled-coil embodiment, thus allowing two differentrandomizing regions to be incorporated simultaneously. This embodimentutilizes non-palindromic BstXI sites on the termini.

In a preferred embodiment, the presentation structure is a sequence thatcontains generally two cysteine residues, such that a disulfide bond maybe formed, resulting in a conformationally constrained sequence. Thisembodiment is particularly preferred when secretory targeting sequencesare used. As will be appreciated by those in the art, any number ofrandom sequences, with or without spacer or linking sequences, may beflanked with cysteine residues. In other embodiments, effectivepresentation structures may be generated by the random regionsthemselves. For example, the random regions may be “doped” with cysteineresidues which, under the appropriate redox conditions, may result inhighly crosslinked structured conformations, similar to a presentationstructure. Similarly, the randomization regions may be controlled tocontain a certain number of residues to confer β-sheet or α-helicalstructures.

In a preferred embodiment, the fusion partner is a targeting sequence.As will be appreciated by those in the art, the localization of proteinswithin a cell is a simple method for increasing effective concentrationand determining function. For example, RAF1 when localized to themitochondrial membrane can inhibit the anti-apoptotic effect of BCL-2.Similarly, membrane bound Sos induces Ras mediated signaling inT-lymphocytes. These mechanisms are thought to rely on the principle oflimiting the search space for ligands, that is to say, the localizationof a protein to the plasma membrane limits the search for its ligand tothat limited dimensional space near the membrane as opposed to the threedimensional space of the cytoplasm. Alternatively, the concentration ofa protein can also be simply increased by nature of the localization.Shuttling the proteins into the nucleus confines them to a smaller spacethereby increasing concentration. Finally, the ligand or target maysimply be localized to a specific compartment, and inhibitors must belocalized appropriately.

Thus, suitable targeting sequences include, but are not limited to,binding sequences capable of causing binding of the expression productto a predetermined molecule or class of molecules while retainingbioactivity of the expression product, (for example by using enzymeinhibitor or substrate sequences to target a class of relevant enzymes);sequences signalling selective degradation, of itself or co-boundproteins; and signal sequences capable of constitutively localizing thecandidate expression products to a predetermined cellular locale,including a) subcellular locations such as the Golgi, endoplasmicreticulum, nucleus, nucleoli, nuclear membrane, mitochondria,chloroplast, secretory vesicles, lysosome, and cellular membrane; and b)extracellular locations via a secretory signal. Particularly preferredis localization to either subcellular locations or to the outside of thecell via secretion.

In a preferred embodiment, the targeting sequence is a nuclearlocalization signal (NLS). NLSs are generally short, positively charged(basic) domains that serve to direct the entire protein in which theyoccur to the cell's nucleus. Numerous NLS amino acid sequences have beenreported including single basic NLS's such as that of the SV40 (monkeyvirus) large T Antigen (Pro Lys Lys Lys Arg Lys Val), Kalderon (1984),et al., Cell, 39:499-509; the human retinoic acid receptor-1 nuclearlocalization signal localization signal (ARRRRP); NFkB p50 (EEVQRKRQKL;Ghosh et al., Cell 62:1019 (1990); NFkB p65 (EEKRKRTYE; Nolan et al.,Cell 64:961 (1991); and others (see for example Boulikas, J. Cell.Biochem. 55(1):32-58 (1994), hereby incorporated by reference) anddouble basic NLS's exemplified by that of the Xenopus (African clawedtoad) protein, nucleoplasmin (Ala Val Lys Arg Pro Ala Ala Thr Lys LysAla Gly Gln Ala Lys Lys Lys Lys Leu Asp), Dingwall, et al., Cell,30:449-458, 1982 and Dingwall, et al., J. Cell Biol., 107:641-849;1988). Numerous localization studies have demonstrated that NLSsincorporated in synthetic peptides or grafted onto reporter proteins notnormally targeted to the cell nucleus cause these peptides and reporterproteins to be concentrated in the nucleus. See, for example, Dingwall,and Laskey, Ann, Rev. Cell Biol., 2:367-390, 1986; Bonnerot, et al.,Proc. Natl. Acad. Sci. USA, 84:6795-6799, 1987; Galileo, et al., Proc.Natl. Acad. Sci. USA, 87:458-462, 1990.

In a preferred embodiment, the targeting sequence is a membraneanchoring signal sequence. This is particularly useful since manyparasites and pathogens bind to the membrane, in addition to the factthat many intracellular events originate at the plasma membrane. Thus,membrane-bound peptide libraries are useful for both the identificationof important elements in these processes as well as for the discovery ofeffective inhibitors. The invention provides methods for presenting therandomized expression product extracellularly or in the cytoplasmicspace; see FIG. 3. For extracellular presentation, a membrane anchoringregion is provided at the carboxyl terminus of the peptide presentationstructure. The randomized epression product region is expressed on thecell surface and presented to the extracellular space, such that it canbind to other surface molecules (affecting their function) or moleculespresent in the extracellular medium. The binding of such molecules couldconfer function on the cells expressing a peptide that binds themolecule. The cytoplasmic region could be neutral or could contain adomain that, when the extracellular randomized expression product regionis bound, confers a function on the cells (activation of a kinase,phosphatase, binding of other cellular components to effect function).Similarly, the randomized expression product-containing region could becontained within a cytoplasmic region, and the transmembrane region andextracellular region remain constant or have a defined function.

Membrane-anchoring sequences are well known in the art and are based onthe genetic geometry of mammalian transmembrane molecules. Peptides areinserted into the membrane based on a signal sequence (designated hereinas ssTM) and require a hydrophobic transmembrane domain (herein TM). Thetransmembrane proteins are inserted into the membrane such that theregions encoded 5′ of the transmembrane domain are extracellular and thesequences 3′ become intracellular. Of course, if these transmembranedomains are placed 5′ of the variable region, they will serve to anchorit as an intracellular domain, which may be desirable in someembodiments. ssTMs and TMs are known for a wide variety of membranebound proteins, and these sequences may be used accordingly, either aspairs from a particular protein or with each component being taken froma different protein, or alternatively, the sequences may be synthetic,and derived entirely from consensus as artificial delivery domains.

As will be appreciated by those in the art, membrane-anchoringsequences, including both ssTM and TM, are known for a wide variety ofproteins and any of these may be used. Particularly preferredmembrane-anchoring sequences include, but are not limited to, thosederived from CD8, ICAM-2, IL-8R, CD4 and LFA-1.

Useful sequences include sequences from: 1) class I integral membraneproteins such as IL-2 receptor beta-chain (residues 1-26 are the signalsequence, 241-265 are the transmembrane residues; see Hatakeyama et al.,Science 244:551 (1989) and von Heijne et al, Eur. J. Biochem. 174:671(1988)) and insulin receptor beta chain (residues 1-27 are the signal,957-959 are the transmembrane domain and 960-1382 are the cytoplasmicdomain; see Hatakeyama, supra, and Ebina et al., Cell 40:747 (1985)); 2)class II integral membrane proteins such as neutral endopeptidase(residues 29-51 are the transmembrane domain, 2-28 are the cytoplasmicdomain; see Malfroy et al., Biochem. Biophys. Res. Commun. 144:59(1987)); 3) type III proteins such as human cytochrome P450 NF25(Hatakeyama, supra); and 4) type IV proteins such as humanP-glycoprotein (Hatakeyama, supra). Particularly preferred are CD8 andICAM-2. For example, the signal sequences from CD8 and ICAM-2 lie at theextreme 5′ end of the transcript. These consist of the amino acids 1-32in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP; Nakauchi et al.,PNAS USA 82:5126 (1985) and 1-21 in the case of ICAM-2(MSSFGYRTLTVALFTLICCPG; Staunton et al., Nature (London) 339:61 (1989)).These leader sequences deliver the construct to the membrane while thehydrophobic transmembrane domains, placed 3′ of the random candidateregion, serve to anchor the construct in the membrane. Thesetransmembrane domains are encompassed by amino acids 145-195 from CD8(PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR; Nakauchi, supra)and 224-256 from ICAM-2 (MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR: Staunton,supra).

Alternatively, membrane anchoring sequences include the GPI anchor,which results in a covalent bond between the molecule and the lipidbilayer via a glycosyl-phosphatidylinositol bond for example in DAF(PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT, with the bolded serine the siteof the anchor; see Homans et al., Nature 333(6170):269-72 (1988), andMoran et al., J. Biol. Chem. 266:1250 (1991)). In order to do this, theGPI sequence from Thy-1 can be cassetted 3′ of the variable region inplace of a transmembrane sequence.

Similarly, myristylation sequences can serve as membrane anchoringsequences. It is known that the myristylation of c-src recruits it tothe plasma membrane. This is a simple and effective method of membranelocalization, given that the first 14 amino acids of the protein aresolely responsible for this function: MGSSKSKPKDPSQR (see Cross et al.,Mol. Cell. Biol. 4(9):1834 (1984); Spencer et al., Science 262:1019-1024(1993), both of which are hereby incorporated by reference). This motifhas already been shown to be effective in the localization of reportergenes and can be used to anchor the zeta chain of the TCR. This motif isplaced 5′ of the variable region in order to localize the construct tothe plasma membrane. Other modifications such as palmitoylation can beused to anchor constructs in the plasma membrane; for example,palmitoylation sequences from the G protein-coupled receptor kinase GRK6sequence (LLQRLFSRQDCCGNCSDSEEELPTRL, with the bold cysteines beingpalmitolyated; Stoffel et al., J. Biol, Chem 269:27791 (1994)); fromrhodopsin (KQFRNCMLTSLCCGKNPLGD; Barnstable et al., J. Mol. Neurosci.5(3):207 (1994)); and the p21 ras 1 protein (LNPPDESGPGCMSCKCVLS; Caponet al., Nature 302:33 (1983)).

In a preferred embodiment, the targeting sequence is a lysozomaltargeting sequence, including, for example, a lysosomal degradationsequence such as Lamp-2 (KFERQ; Dice, Ann. N.Y. Acad. Sci. 674:58(1992); or lysosomal membrane sequences from Lamp-1(MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI, Uthayakumar et al., Cell. Mol.Biol. Res. 41:405 (1995)) or Lamp-2(LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF, Konecki et la., Biochem. Biophys.Res. Comm. 205:1-5 (1994), both of which show the transmembrane domainsin italics and the cytoplasmic targeting signal underlined).

Alternatively, the targeting sequence may be a mitrochondriallocalization sequence, including mitochondrial matrix sequences (e.g.yeast alcohol dehydrogenase Ill; MLRTSSLFTRRVQPSLFSRNILRLQST; Schatz,Eur. J. Biochem. 165:1-6 (1987)); mitochondrial inner membrane sequences(yeast cytochrome c oxidase subunit IV; MLSLRQSIRFFKPATRTLCSSRYLL;Schatz, supra); mitochondrial intermembrane space sequences (yeastcytochrome c1;MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYADSLTAEAMTA;Schatz, supra) or mitochondrial outer membrane sequences (yeast 70 kDouter membrane protein; MKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQQRGKK;Schatz, supra).

The target sequences may also be endoplasmic reticulum sequences,including the sequences from calreticulin (KDEL; Pelham, Royal SocietyLondon Transactions B; 1-10 (1992)) or adenovirus E3/19K protein(LYLSRRSFIDEKKMP; Jackson et al., EMBO J. 9:3153 (1990).

Furthermore, targeting sequences also include peroxisome sequences (forexample, the peroxisome matrix sequence from Luciferase; SKL; Keller etal., PNAS USA 4:3264 (1987)); farnesylation sequences (for example, P21H-ras 1; LNPPDESGPGMSCKCVLS, with the bold cysteine farnesylated; Capon,supra); geranylgeranylation sequences (for example, protein rab-5A;LTEPTQPTRNQCCSN, with the bold cysteines geranylgeranylated; Farnsworth,PNAS USA 91:11963 (1994)); or destruction sequences (cyclin B1;RTALGDIGN; Klotzbucher et al., EMBO J. 1:3053 (1996)).

In a preferred embodiment, the targeting sequence is a secretory signalsequence capable of effecting the secretion of the candidate translationproduct. There are a large number of known secretory signal sequenceswhich are placed 5′ to the variable peptide region, and are cleaved fromthe peptide region to effect secretion into the extracellular space.Secretory signal sequences and their transferability to unrelatedproteins are well known, e.g., Silhavy, et al. (1985) Microbiol. Rev.49, 398-418. This is particularly useful to generate a peptide capableof binding to the surface of, or affecting the physiology of, a targetcell that is other than the host cell, e.g., the cell expressing thepeptide. In a preferred approach, a fusion product is configured tocontain, in series, secretion signal peptide-presentationstructure-randomized expression product region-presentation structure.In this manner, target cells grown in the vicinity of cells caused toexpress the library of peptides, are bathed in secreted peptide. Targetcells exhibiting a physiological change in response to the presence of apeptide, e.g., by the peptide binding to a surface receptor or by beinginternalized and binding to intracellular targets, and the secretingcells are localized by any of a variety of selection schemes and thepeptide causing the effect determined. Exemplary effects includevariously that of a designer cytokine (i.e., a stem cell factor capableof causing hematopoietic stem cells to divide and maintain theirtotipotential), a factor causing cancer cells to undergo spontaneousapoptosis, a factor that binds to the cell surface of target cells andlabels them specifically, etc.

Suitable secretory sequences are known, including signals from IL-2(MYRMQLLSCIALSLALVTNS; Villinger et al., J. Immunol. 155:3946 (1995)),growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT; Roskam et al., NucleicAcids Res. 7:30 (1979)); preproinsulin (MALWMRLLPLLALLALWGPDPAAAFVN;Bell et al., Nature 284:26 (1980)); and influenza HA protein(MKAKLLVLLYAFVAGDQI; Sekiwawa et al., PNAS 80:3563)), with cleavagebetween the non-underlined-underlined junction. A particularly preferredsecretory signal sequence is the signal leader sequence from thesecreted cytokine IL-4, which comprises the first 24 amino acids of IL-4as follows: MGLTSQLLPPLFFLLACAGNFVHG.

In a preferred embodiment, the fusion partner is a rescue sequence. Arescue sequence is a sequence which may be used to purify or isolateeither the candidate agent or the nucleic acid encoding it. Thus, forexample, peptide rescue sequences include purification sequences such asthe His₆ tag for use with Ni affinity columns and epitope tags fordetection, immunoprecipitation or FACS (fluoroscence-activated cellsorting). Suitable epitope tags include myc (for use with thecommercially available 9E10 antibody), the BSP biotinylation targetsequence of the bacterial enzyme BirA, flu tags, lacZ, and GST.

Alternatively, the rescue sequence may be a unique oligonucleotidesequence which serves as a probe target site to allow the quick and easyisolation of the retroviral construct, via PCR, related techniques, orhybridization.

In a preferred embodiment, the fusion partner is a stability sequence toconfer stability to the candidate bioactive agent or the nucleic acidencoding it. Thus, for example, peptides may be stabilized by theincorporation of glycines after the initiation methionine (MG or MGGO),for protection of the peptide to ubiquitination as per Varshaysky'sN-End Rule, thus conferring long half-life in the cytoplasm.

Similarly, two prolines at the C-terminus impart peptides that arelargely resistant to carboxypeptidase action. The presence of twoglycines prior to the prolines impart both flexibility and preventstructure initiating events in the di-proline to be propagated into thecandidate peptide structure. Thus, preferred stability sequences are asfollows: MG(X)_(n)GGPP, where X is any amino acid and n is an integer ofat least four.

In one embodiment, the fusion partner is a dimerization sequence. Adimerization sequence allows the non-covalent association of one randompeptide to another random peptide, with sufficient affinity to remainassociated under normal physiological conditions. This effectivelyallows small libraries of random peptides (for example, 10⁴) to becomelarge libraries if two peptides per cell are generated which thendimerize, to form an effective library of 10⁸ (10⁴×10⁴). It also allowsthe formation of longer random peptides, if needed, or more structurallycomplex random peptide molecules. The dimers may be homo- orheterodimers.

Dimerization sequences may be a single sequence that self-aggregates, ortwo sequences, each of which is generated in a different retroviralconstruct. That is, nucleic acids encoding both a first random peptidewith dimerization sequence 1, and a second random peptide withdimerization sequence 2, such that upon introduction into a cell andexpression of the nucleic acid, dimerization sequence 1 associates withdimerization sequence 2 to form a new random peptide structure.

Suitable dimerization sequences will encompass a wide variety ofsequences. Any number of protein-protein interaction sites are known. Inaddition, dimerization sequences may also be elucidated using standardmethods such as the yeast two hybrid system, traditional biochemicalaffinity binding studies, or even using the present methods.

The fusion partners may be placed anywhere (i.e. N-terminal, C-terminal,internal) in the structure as the biology and activity permits.

In a preferred embodiment, the fusion partner includes a linker ortethering sequence, as generally described in PCT US 97/01019, that canallow the candidate agents to interact with potential targetsunhindered. For example, when the candidate bioactive agent is apeptide, useful linkers include glycine-serine polymers (including, forexample, (GS)_(n), (GSGGS)_(n) and (GGGS)_(n), where n is an integer ofat least one), glycine-alanine polymers, alanine-serine polymers, andother flexible linkers such as the tether for the shaker potassiumchannel, and a large variety of other flexible linkers, as will beappreciated by those in the art. Glycine-serine polymers are preferredsince both of these amino acids are relatively unstructured, andtherefore may be able to serve as a neutral tether between components.Secondly, serine is hydrophilic and therefore able to solubilize whatcould be a globular glycine chain. Third, similar chains have been shownto be effective in joining subunits of recombinant proteins such assingle chain antibodies.

In addition, the fusion partners, including presentation structures, maybe modified, randomized, and/or matured to alter the presentationorientation of the randomized expression product. For example,determinants at the base of the loop may be modified to slightly modifythe internal loop peptide tertiary structure, which maintaining therandomized amino acid sequence.

In a preferred embodiment, combinations of fusion partners are used.Thus, for example, any number of combinations of presentationstructures, targeting sequences, rescue sequences, and stabilitysequences may be used, with or without linker sequences.

Thus, candidate agents can include these components, and may then beused to generate a library of fragments, each containing a differentrandom nucleotide sequence that may encode a different peptide. Theligation products are then transformed into bacteria, such as E. coli,and DNA is prepared from the resulting library, as is generally outlinedin Kitamura, PNAS USA 92:9146-9150 (1995), hereby expressly incorporatedby reference.

Delivery of the library DNA into a retroviral packaging system resultsin conversion to infectious virus. Suitable retroviral packaging systemcell lines include, but are not limited to, the Bing and BOSC23 celllines described in WO 94/19478; Soneoka et al., Nucleic Acid Res.23(4):628 (1995); Finer et al., Blood 83:43 (1994); Pheonix packaginglines such as PhiNX-eco and PhiNX-ampho, described below; 292T+gag-poland retrovirus envelope; PA317; and cell lines outlined in Markowitz etal., Virology 167:400 (1988), Markowitz et al., J. Virol. 62:1120(1988), Li et al., PNAS USA 93:11658 (1996), Kinsella et al., Human GeneTherapy 7:1405 (1996), all of which are incorporated by reference.Preferred systems include PhiNX-eco and PhiNX-ampho or similar celllines, disclosed in PCT US97/01019.

When the cells are not replicating, other viral vectors may be used,including adenoviral vectors, feline immunoviral (FIV) vectors, etc.

In a preferred embodiment, when the candidate agent is introduced to thecells using a viral vector, the candidate peptide agent is linked to adetectable molecule, and the methods of the invention include at leastone expression assay. An expression assay is an assay that allows thedetermination of whether a candidate bioactive agent has been expressed,i.e. whether a candidate peptide agent is present in the cell. Thus, bylinking the expression of a candidate agent to the expression of adetectable molecule such as a label, the presence or absence of thecandidate peptide agent may be determined. Accordingly, in thisembodiment, the candidate agent is operably linked to a detectablemolecule. Generally, this is done by creating a fusion nucleic acid. Thefusion nucleic acid comprises a first nucleic acid encoding thecandidate bioactive agent (which can include fusion partners, asoutlined above), and a second nucleic acid encoding a detectablemolecule. The terms “first” and “second” are not meant to confer anorientation of the sequences with respect to 5′-3′ orientation of thefusion nucleic acid. For example, assuming a 5′-3′ orientation of thefusion sequence, the first nucleic acid may be located either 5′ to thesecond nucleic acid, or 3′ to the second nucleic acid. Preferreddetectable molecules in this embodiment include, but are not limited to,fluorescent proteins, including GFP, YFP, BFP and RFP, with the formerbeing especially preferred.

In general, the candidate agents are added to the cells (eitherextracellularly or intracellularly, as outlined above) under reactionconditions that favor agent-target interactions. Generally, this will bephysiological conditions. Incubations may be performed at anytemperature which facilitates optimal activity, typically between 4 and40° C. Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high through put screening.Typically between 0.1 and 1 hour will be sufficient. Excess reagent isgenerally removed or washed away.

A variety of other reagents may be included in the assays. These includereagents like salts, neutral proteins, e.g. albumin, detergents, etcwhich may be used to facilitate optimal protein-protein binding and/orreduce non-specific or background interactions. Also reagents thatotherwise improve the efficiency of the assay, such as proteaseinhibitors, nuclease inhibitors, anti-microbial agents, etc., may beused. The mixture of components may be added in any order that providesfor detection. Washing or rinsing the cells will be done as will beappreciated by those in the art at different times, and may include theuse of filtration and centrifugation. When second labeling moieties(also referred to herein as “secondary labels”) are used, they arepreferably added after excess non-bound target molecules are removed, inorder to reduce non-specific binding; however, under some circumstances,all the components may be added simultaneously.

In a preferred embodiment, the cells are sorted usingfluorescent-activated cell sorting (FACS). In the invention herein, cellcycle regulation is evaluated by multiple parameters which results inreduced background and greater specificity. In contrast, FACS has beenused in the past to evaluate two different or unrelated characteristicsat the same time which identifies cells having those twocharacteristics, but does not reduce the background for the combinedcharacteristics.

Thus, the cells are sorted or enriched in a FACS on the basis of one ormore of the assays, including a cell viability assay, a proliferationassay, a cell phase assay, and (when candidate agents are expressed withdetectable moieties) an expression assay. The results from one or moreof these assays are compared to cells that were not exposed to thecandidate bioactive agent, or to the same cells prior to introduction ofthe candidate agent. Alterations in these results can indicate that saidagent modulates cell cycle regulation.

A strength of the present invention is that a library of candidateagents may be tested in a library of cells, because the present methodsallow single cell sorting, with extremely high specificity, such thatvery rare events may be detected. The use of multiple laser paths allowssort accuracy of 1 in 10⁶ with better than 70% accuracy.

In addition, the present invention can, in addition to theidentification of multiple cell cycle regulation properties, be combinedwith the identification of other cellular characteristics. For example,parameters of general cellular health can be determined and selected forby using i.e., dye Indo-1 indicating a calcium response. Other cellularparameters which are routinely identified by the skilled artisan includebut are not limited to: cell size, cell shape, redox state, DNA content,nucleic acid sequence, chromatin structure, RNA content, total protein,antigens, lipids, surface proteins, intracellular receptors, oxidativemetabolism, DNA synthesis and degradation and intracellular pH.

In a preferred embodiment, each of the measurements is determinedsimultaneously from an individual cell as it passes through the beampaths of multiple lasers. Alternatively, the measurements are donesequentially. By using more than one parameter to detect cell cycleregulation or alterations in cell cycle regulation, background isreduced and specificity is increased. The cells meeting the parametersof the desired properties can be physically sorted from cells notmeeting the desired parameters or they can be identified by theirpercentage in the cell population.

In general, K_(D) s of ≦1 μM are preferred, to allow for retention ofbinding in the presence of the shear forces present in FACS sorting. Ina preferred embodiment, the cells are sorted at very high speeds, forexample greater than about 5,000 sorting events per sec, with greaterthan about 10,000 sorting events per sec being preferred, and greaterthan about 25,000 sorting events per second being particularlypreferred, with speeds of greater than about 50,000 to 100,000 beingespecially preferred.

Cells processed for stimulation and staining are generally taken up inbuffer and filtered prior to cytometry. Cells can be analyzed using aFACSCAN (Becton Dickinson Inc., laser line 488 nm) or a Mo-Flo(Cytomation, Inc., laser lines 350 nM broadband (UV), 488 nm, and 647nm) Cytometer. Cells are sorted, if desired, using the Mo-Flo.

Wherein the cells are analyzed by microscopy, cells post stimulation orstaining are generally mounted onto glass slides and coverslipped; theseare directly visualized by brightfield and fluorescence microscopy on aninverted microscope (i.e., TE300, Nikon) using standard BFP, FITC, orTRITC (for example) filter sets. Images can also be obtained using aninverted confocal scanning microscope (Zeiss, Inc, Bio-Rad, Inc.) usingstandard FITC and TRITC (for example) filter sets.

The sorting results in a population of cells having the desiredproperties. In a preferred embodiment, the parameters are set toidentify at least one candidate bioactive agent that modulates cellcycle regulation.

In a preferred embodiment, the bioactive agent is characterized. Thiswill proceed as will be appreciated by those in the art, and generallyincludes an analysis of the structure, identity, binding affinity andfunction of the agent. Generally, once identified, the bioactive agentis resynthesized and combined with the target cell to verify the cellcycle regulation modulation under various conditions and in the presenceor absence of other various agents. The bioactive can be prepared in atherapeutically effective amount to modulate cell cycle regulation andcombined with a suitable pharmaceutical carrier.

In a preferred embodiment, the cell populations can be subjected tovarious experimental conditions, with and without the candidate agents.Changes in conditions include but are not limited to changes in pH,temperature, buffer or salt concentration, etc. In a preferredembodiment, the pH is changed, generally by increasing or decreasing thepH, usually by from about 0.5 to about 3 pH units. Alternatively, thetemperature is altered, with increases or decreases of from about 5° C.to about 30° C. being preferred. Similarly, the salt concentration maybe modified, with increases or decreases of from about 0.1 M to about 2M being preferred.

It is understood by the skilled artisan that the steps of the assaysprovided herein can vary in order. It is also understood, however, thatwhile various options (of compounds, properties selected or order ofsteps) are provided herein, the options are also each providedindividually, and can each be individually segregated from the otheroptions provided herein. Moreover, steps which are obvious and known inthe art that will increase the sensitivity of the assay are intended tobe within the scope of this invention. For example, there may beadditionally washing steps, or segregation, isolation steps. Moreover,it is understood that in some cases detection is in the cells, but canalso take place in the media, or vice versa.

In a preferred embodiment, the cellular phenotype is exocytosis, and themethods and compositions of the invention are directed to the detectionof alterations in exocytosis, again using a FACS machine. There are anumber of parameters that may be evaluated or assayed to allow thedetection of alterations in exocytotic pathways, including, but notlimited to, light scattering, fluorescent dye uptake, fluorescent dyerelease, granule exposure, surface granule enzyme activity, and thequantity of granule specific proteins. By assaying or measuring one ormore of these parameters, it is possible to detect not only alterationsin exocytosis, but alterations of different steps of the exocytoticpathway. In addition, multiparameter analysis also reduces thebackground, or “false positives”, that are detected. In this manner,rapid, accurate screening of candidate agents may be performed toidentify agents that modulate exocytosis.

In a preferred embodiment, the invention provides methods for screeningfor alterations in exocytosis of a population of cells. By “alteration”or “modulation” in the context of exocytosis is meant a decrease or anincrease in the amount of exocytosis in one cell compared to anothercell or in the same cell under different conditions. The measurementscan be determined wherein all of the conditions are the same for eachmeasurement, or under various conditions, with or without bioactiveagents, or at different stages of the exocytic process. For example, ameasurement of exocytosis can be determined in a cell population whereina candidate bioactive agent is present and wherein the candidatebioactive agent is absent. In another example, the measurements ofexocytosis are determined wherein the condition or environment of thepopulations of cells'differ from one another. For example, the cells maybe evaluated in the presence or absence of physiological signals, suchas exocytic inducers (i.e, Ca⁺⁺, ionomycin, etc.), hormones, antibodies,peptides, antigens, cytokines, growth factors, action potentials, orother cells (i.e. cell-cell contacts). In another example, themeasurements of exocytosis are determined at different stages of theexocytic process. In yet another example, the measurements of exocytosisare taken wherein the conditions are the same, and the alterations arebetween one cell or cell population and another cell or cell population.

By a “population of cells” herein is meant a sample of cells as definedabove. In this embodiment, the cells are preferably (but not required)to be rapidly growing, retrovirally infectable, and compatible with dyesand antibodies. Preferred cell types for use in this embodiment,include, but are not limited to, mast cells, neurons, adrenal chromaffincells, basophils, endocrine cells including pancreatic β-cells,pancreatic acinar cells including exocrine cells, neutrophils,monocytes, lymphocytes, mammary cells, sperm, egg cells and PMNleukocytes, endothelial cells, adipocytes, and muscle cells.

The exocytotic methods comprise sorting the cells in a FACS machine byassaying for alterations in at least three of the properties selectedfrom the group consisting of light scattering, fluorescent dye uptake,fluorescent dye release, granule exposure, surface granule enzymeactivity, and the quantity of granule specific proteins. In a preferredembodiment, each of the measurements is determined simultaneously froman individual cell as it passes through the beam paths of multiplelasers. Alternatively, the measurements are done sequentially. By usingmore than one parameter to detect exocytosis or alterations inexocytosis, background is reduced and specificity is increased. Thecells meeting the parameters of the desired properties can be physicallysorted from cells not meeting the desired parameters or they can beidentified by their percentage in the cell population.

In a preferred embodiment, changes in light scattering are assayed todetermine alterations in exocytosis in a population of cells. Whenviewed in the FACS, cells have particular characteristics as measured bytheir forward and 90 degree (side) light scatter properties. Thesescatter properties represent the size, shape and granule content of thecells. Upon activation of the cells with a pro-exocytic stimulus, boththe forward and side scatter properties of the cells changesconsiderably. These properties account for two parameters to be measuredas a readout for the exocytic event. These properties change inproportion to the extent of exocytosis of the cells and depend on thetime course of the exocytic events as well. Alterations in the intensityof light scattering, or the cell-refractive index indicate alterationsin exocytosis either in the same cell at different times, or compared tothe same cell under different conditions or with candidate bioactiveagents present or absent, or compared to different cells or cellpopulations.

In one embodiment provided herein, a cell population is combined with anagent which is known to stimulate exocytosis and the light scatteringproperties are determined. Cells having light scattering propertiesindicating the desirable exocytic activity can be identified and/orsorted. Exocytic activity as used herein includes lack of activity. In apreferred embodiment, candidate bioactive agents are combined with thecell population prior to or with the exocytic stimulus, as is more fullyoutlined below. In this embodiment, where light scattering propertiesdiffer as between a) a cell population combined with a known exocyticstimulus and a candidate bioactive agent, and b) a cell populationcombined with a known exocytic stimulus wherein the candidate bioactiveagent is absent, it can be determined that the candidate bioactive agentmodulates exocytosis. It may also be desirable in some cases to includean inhibitor of exocytosis or to exclude the exocytic stimulus toidentify bioactive agents which induce exocytosis. Preferably, lightscattering properties are measured in combination with at least one, andpreferably two other properties which indicate exocytosis activity.General methodologies for light scattering measurements are furtherdescribed in Perretti, et al., J. Pharmacol. Methods, 23(3):187-194(1990) and Hide et al., J. Cell Biol., 123(3):585-593 (1993), bothincorporated herein by reference. In general, changes of at least about5% from baseline are preferred, with at least about 25% being morepreferred, at least about 50% being particularly preferred, and at leastabout 75 to 100% being especially preferred. Baseline in this casegenerally means the light scatter properties of the cells prior toexocytotic stimulation. In each case provided herein, the baseline mayalso be set for any control parameter. For example, the baseline may beset at the exocytosis measurement of a particular cell, a similar cellunder different conditions, or at a particular time point duringexocytosis.

In another preferred embodiment, changes in fluorescent dye uptake areevaluated. Preferred fluorescent dyes include styryl dyes, whichindicate exocytosis activity in relation to endocytosis, sometimesreferred to as coupled endocytosis. The theory behind coupledendocytosis is that cells undergoing exocytosis must also undergoendocytosis in order to maintain cell volume and membrane integrity.Thus, upon exocytic stimulation, endocytosis is also increased,providing an indirect measurement of exocytosis by quantifying theamount of styryl dye uptake.

In an embodiment provided herein, the cells are bathed in a solution ofstyryl dye and stimulated with a pro-exocytic stimulus and the dye isquantitated. Preferably, after exocytic stimulation, the cells are spundown, aspirated and resuspended in fresh buffer. In a preferredembodiment, a candidate bioactive agent is combined with the cells asdescribed herein. In some cases, the candidate bioactive agent can becombined with the cells with an inhibitor of exocytosis or without thepro-exocytic stimulus. Preferably, a pro-exocytic stimulus is added tothe cell population which results in a dramatic increase in thefluorescence signal of the dye. The increased cell associated signal isdue to coupled endocytosis of the styryl dye and is proportional to theexocytic response in both time and intensity. Conversely, the signal isnot increased wherein exocytosis is inhibited or is not induced.Alterations are determined by measuring the fluorescence ateither-different time points or in different cell populations, andcomparing the determinations to one another or to standards. In general,changes of at least about 50% from baseline are preferred, with changesof at least about 75%-100% being more preferred, changes of at leastabout 250% being particularly preferred, and changes of at least about1000-2000% being especially preferred. Baseline in this case means thestyryl dye uptake of cells prior to exocytic stimulation.

Preferred styryl dyes include, but are not limited to FM1-43, FM4-64,FM14-68, FM2-10, FM4-84, FM1-84, FM14-27, FM14-29, FM3-25, FM3-14,FM5-55, RH414, FM6-55, FM10-75, FM1-81, FM9-49, FM4-95, FM4-59, FM9-40,and combinations thereof. Preferred dyes such as FM1-43 are only weaklyfluorescent in water but very fluorescent when associated with amembrane, such that dye uptake is readily discernable. Suitable dyes areavailable commercially, i.e., Molecular Probes, Inc., of Eugene, Oreg.,“Handbook of Fluorescent Probes and Research Chemicals”, 6th Edition,1996, particularly, Chapter 17, and more particularly, Section 2 ofChapter 17, (including referenced related chapter), hereby incorporatedherein by reference. Preferably, the dyes are provided in a solutionwherein the dye concentration is about 25 to 1000-5000 nM, with fromabout 50 to about 1000 nM being preferred, and from about 50 to 250being particularly preferred. The use of styryl dyes is furtherdescribed in Betz, et al., Current Opinion in Neurobiology, 6:365-371(1996) also incorporated herein by reference. Preferably, fluorescentdye uptake is measured in combination with at least one, and preferablytwo other indicators of exocytosis activity.

In another preferred embodiment, changes in fluorescent dye release areevaluated. The present invention is in part directed to the discoverythat low pH concentration dyes, which are normally used to stainlysozomes, also low pH stain exocytic granules. Generally, these dyescan be taken up by the cells passively and concentrate in granules:however, the cells can be induced to take up the dye, i.e., by coupledendocytosis. In a preferred embodiment, a cell population is bathed in alow pH concentration dye such that the dye is taken up by the cells. Thecells are preferably washed. The cells can be exposed to a pro-exocyticstimulus and/or inhibitor. In a preferred embodiment, a candidatebioactive agent is combined with the cell population and preferably, thepro-exocytic stimulus. Fluorescence is evaluated. Changes in fluorescentdye release between cells or at different time points in the same cellindicate alterations in exocytosis. Preferably, the alterations arebetween cells, and most preferably, between cells having differentbioactive agents added thereto. Changes of at least about 5% frombaseline are preferred, with at least about 25% being more preferred, atleast about 50% being particularly preferred and at least about 100%being especially preferred. Baseline in this case means the amount ofdye in the cells prior to stimulation.

In this embodiment, low pH concentration dyes are preferred. Such low pHconcentration dyes include but are not limited to acridine orange.LYSOTRACKER™ red, LYSOTRACKER™ green, and LYSOTRACKER™ blue. Such dyesare commercially available, i.e., from Molecular Probes, supra,particularly including Chapter 17, Section 4 of Chapter 17, andreferenced “related chapters”, i.e., Chapter 23. In preferredembodiments, the dyes are administered in a solution wherein the dye isa concentration of about 50 nM to about 25 μM, with from about 5 μM toabout 25 μM being preferred, and from about 1 to 5 μM being particularlypreferred. The use of low pH concentration dyes is generally described(in regards to lysozome studies) in Haller, et al., Cell Calcium,19(2):157-165 (1996), hereby incorporated herein by reference.

In an alternative embodiment wherein changes in fluorescent dye releaseare evaluated, the fluorescence released into the supernatant isevaluated. In this embodiment, either styryl dyes, which reversiblylabel endocytosed membranes, or low pH concentration dyes are used. Inthis embodiment, a cell population is bathed in dye such that the dye istaken up into the cells passively or by induction. The cells are thenpreferably washed. The cells can be exposed to a pro-exocytic stimulusand/or inhibitor, and optionally, a candidate bioactive agent. The cellswhich are exposed to a pro-exocytic stimulus will release the dye intothe extracellular medium. The fluorescence in the medium can be measuredor detected. This process is sometimes referred to as destaining thecells. Optionally, an agent for improving and facilitating the detectionof the dye in the medium can be added. For example, micelle-formingdetergents such as3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)increase the fluorescence and thereby allow detection of small amountsof exocytosis activity. Changes in the release of dye will indicatealterations in exocytosis in the same cell, between cells, and mostpreferably, between cells having different bioactive agents addedthereto. In general, changes of at least about 5% from baseline arepreferred, with at least about 25% being more preferred, with at leastabout 50% being particularly preferred and at least about 100% beingespecially preferred. Baseline in this case means the release of dyeprior to exocytotic stimulus. Preferably, dye release when measured inthe media is combined with the evaluation of at least one otherexocytosis indicator.

In a preferred embodiment, changes in granule exposure are determined.The granules are exposed to the media during exocytosis, i.e., thegranules fuse with the cell membrane and expose/release their contents.Therefore, granule exposure is indicative of exocytic activity, and itsabsence is indicative that exocytosis has not been induced, or has beeninhibited. Preferably, granule exposure is detected by a detectableagent which specifically bind to granules. An example of a detectableagent used herein is annexin V, a member of a protein family whichdisplays specific binding to phospholipid (phosphotidylserine) in adivalent ion dependent manner. This protein has been widely used for themeasurement of apoptosis (programmed cell death) as cell surfaceexposure of phosphatidylserine is a hallmark early signal of thisprocess. Surprisingly, it has been determined herein that annexin Vspecifically binds to exocytic granules when they are exposed at thecell surface during the secretory process; granules internal to the cellare unlabeled. This property of annexin V is used herein to create asingle exocytosis assay based on its exocytosis dependent binding. Uponexocytic stimulation of cells, the cells show an increase in annexinbinding and fluorescent signal in proportion in both time and intensityto the exocytic response.

In this embodiment, annexin is labelled, either directly or indirectly,and combined with a cell population. Annexin is commercially available,i.e., from PharMingen, San Diego, Calif., or Caltag Laboratories,Millbrae, Calif. Preferably, the annexin is provided in a solutionwherein the annexin is in a concentration of about 100 ng/ml to about500 ng/ml, more preferably, about 500 ng/ml to about 1 μg/ml, and mostpreferably, from about 1 μg/ml to about 5 μg/ml. In a preferredembodiment, the annexin is directly labelled; for example, annexin maybe labelled with a fluorochrome such as fluorecein isothiocyanate(FITC), Alexa dyes, TRITC, AMCA, APC, tri-color, Cy-5, and others knownin the art or commercially available. In an alternate preferredembodiment, the annexin is labelled with a first label, such as a haptensuch as biotin, and a secondary fluorescent label is used, such asfluorescent streptavidin. Other first and second labelling pairs can beused as will be appreciated by those in the art.

In the preferred embodiment, the cells are subjected to conditions thatnormally cause exocytosis. Optionally, a candidate bioactive agent isadded to the cells. In some cases, it may be desirable to include aninhibitor of exocytosis to determine whether the candidate agent canreverse the inhibition, or to add the candidate bioactive agent withoutan exocytic stimulus to determine whether the agent induces exocytosis.The cells are preferably washed and fluorescence is detected in themicroscope or on the flowcytometer. Alterations in the detection ofannexin binding indicates alterations in exocytosis in the same cell, orbetween different cells, with or with the same conditions and/or agentscombined therewith. In general, changes of at least about 25% frombaseline are preferred, with at least about 50% being more preferred, atleast about 100 being particularly preferred and at least about 500%being especially preferred. Baseline in this case means the amount ofannexin binding prior to exocytic stimulation.

In another preferred embodiment, granule exposure is detected by acationic dye such as berberine or ruthenium red. Such cationic dyesspecifically stain secreting granules. Thus, when exocytosis occurs, andsecreting granules are exposed at the cell surface, an increase influorescence can be detected. In a preferred embodiment, the cationicdye is combined with a cell population in the presence or absence of anexocytic stimulus and/or inhibitor, and optionally, in the presence orabsence of a candidate bioactive agent. In a particularly preferredembodiment, the berberine is combined with a cell and an exocyticstimulus and a candidate bioactive agent to determine whether thecandidate bioactive agent can modulate the exocytic activity.Preferably, the cells are washed and then fluorescence is determined. Inpreferred embodiments, cationic dye evaluation is combined withevaluation of at least one other indicator of exocytosis. The dye iscombined with the cells as is known in the art. General methodologiesdescribing berberine are described in Berlin and Enerback, Int. Arch.Allergy Appl. Immunol., 73(3):256-262 (1984) hereby incorporated byreference. In general, changes of at least about 5% from baseline arepreferred, with at least about 25% being more preferred, at least about50% being particularly preferred, and at least about 100% beingespecially preferred. Baseline in this case means the amount of dyebinding prior to stimulation.

Similarly, Con A-FITC can be used, as it binds to the carbohydrate ongranule proteins, in a manner similar to those outlined herein.

In another preferred embodiment, changes in surface granule enzymeactivity is determined. Secretory granules contain enzymes such asproteases and glycosidases which are released as part of the exocyticprocess. Frequently, these enzymes are inactive within the granule, dueto the low pH, but upon exposure to the extracellular media atphysiological pH, they become activated. These enzyme activities can bemeasured using chromogenic or fluorogenic substrates as components ofthe extracellular media. This allows detection of exocytic cells invarying approaches.

In one embodiment, sometimes called herein the population based enzymeassay, the generation of signal via cleavage of a chromogenic orfluorogenic substrate can be quantified in the media. That is, theamount of detectable reaction product in the media is related to theamount of enzyme present, and thus to the amount of exocytosis. In thisembodiment, it is the media, not the cells, that becomes detectable.

In a preferred embodiment, cells are subjected to an exocytic stimulus,and optionally, a candidate bioactive agent. The chromogenic orfluorogenic substrate is added to the media, and changes in the signalare evaluated, as the enzymes cleave the extracellular substrates.

In an alternate preferred embodiment, sometimes called herein “in situenzymology assay”, fluorogenic substrates that precipitate upon cleavageare used. That is, upon exocytosis a considerable amount of enzymeactivity remains cell/granule associated and can be visualized usingfluorescent substrates which precipitate at the site of activity. Forexample, substrates for glucuronidase, such as ELF-97 glucuronide,precipitate on exocytosing cells, but not resting cells, and thus thecells can show increased fluorescence. The fluorescence is a directmeasurement of exocytosis and is pH dependent reflecting the pH optimaof the exocytosed enzyme. This method also provides a method ofdistinguishing different subtypes of granules based on their enzymeprofile.

In a preferred embodiment, the cell population is subjected to anexocytic stimulus and then incubated with a detectable substrate. Acandidate bioactive agent is optionally added. The cells are washed andthen viewed in the microscope or flowcytometer.

Preferred granule enzymes include but are not limited to chymase,tryptase, arylsulfatase A, beta-hexosaminidase, beta-glucuronidase, andbeta-D-galactosidase. Substrates include ELF-97 glucuronide, N-acetylbeta-D glucoronide, ELF-97 coupled to peptides, etc., many of which arecommercially available, i.e., from Molecular Probes, supra, particularChapter 10, more particularly Section 2 of Chapter 10, and referenced“related chapters”.

By detectable substrate is meant that the substrate comprises afluorescent molecule as further described herein, or can be detectedwith a fluorescent molecule specific for the substrate or cleavedsubstrate, i.e., a fluorescent antibody. In a preferred embodiment, thesubstrate comprises a detectable molecule formed of two fluorescentproteins, i.e., blue and green fluorescent protein (BFP and GFP), andother similar molecules. As is known in the art, constructs of GFP andBFG that hold these two proteins in close proximity allow fluorescenceresonance energy transfer (FRET). That is, the excitation spectra of theGFP overlaps the emission spectra of the BFP. Accordingly, exciting theBFP results in GFP emission. If a protease cleavage site is engineeredbetween the GFP and BFP to form a “FRET construct”, upon exposure of theFRET construct to an active protease which cleaves the construct, theGFP and BFP molecules separate. Thus, exciting the GFP results in BFPemission and loss of BFP emission.

Preferably, the protease dependent cleavage site inserted between twofluoroscent proteins of the FRET construct is specific for a granulespecific enzyme. Thus, the FRET construct can be used for detectinggranule specific proteases specific for the cleavage site of the FRETconstruct. In this embodiment, the protease substrate that is combinedwith the cells or media includes the FRET construct. The FRET systemallows for detection of the detectable molecule in its cleaved anduncleaved state, and distinguishes between the two. The system isfurther described in Xu et al., Nucleic Acid Res. 26(8):2034 (1998); andMiyawaki et al., Nature 388(6645):882-887 (1997), both of which areincorporated by reference.

The amount of substrate added to the cells or media will depend in parton the enzyme's specific activity and the substrate itself, butgenerally is about 250 nM to about 1 mM, from about 1 μM to about 100 μMbeing preferred, and from about 1 μM to about 10 μM being particularlypreferred. In general, changes of at least about 5% from baseline arepreferred, with at least about 25% being preferred, at least about 100%being particularly preferred and at least about 1000% being especiallypreferred. Baseline in this case means the amount of substrate cleavageprior to induction of exocytosis.

In a preferred embodiment, changes in the quantity of granule specificproteins are determined. Secretory granules contain proteins which arespecifically targeted to the granule compartment due to specificproperties of these proteins. Upon exocytic induction, the granulespecific proteins are exposed to the surface and detected.

In a preferred embodiment, detectable granule specific proteins arecombined with a population of cells and subjected to conditions known toinduce exocytosis. Optionally, a bioactive candidate is combined withthe cell population and detectable granule specific protein and thegranule specific protein is detected. Granule specific proteins includebut are not limited to VAMP and synaptotagmin. Also included within thedefinition of granule specific proteins are the mediators releasedduring exocytosis, including, but not limited to, serotonin, histamine,heparin, hormones, etc.

The quantification of the granule proteins may be done in several ways.In one embodiment, labelled antibodies, (such as fluoroscentantibodies), to granule specific proteins are used. In anotherembodiment, the cells are engineered to contain fusion proteinscomprising a granule protein and a detectable molecule. In a preferredembodiment, a detectable molecule is added to the cells for detection.For example, either directly or indirectly labelled antibodies can beused. A preferred embodiment uses a first labelled antibody, withfluorescent labels preferred. Another embodiment uses a first and secondlabel, for example, a labelled secondary antibody. Generally, thisembodiment may use any agent that will specifically bind to the granuleprotein or compound that can be either directly or indirectly labelled.

In a preferred embodiment the labels are engineered into the cells. Forexample, recombinant proteins are introduced to the cell populationwhich are fusion proteins of a granule specific protein and a detectablemolecule. This is generally done by transforming the cells with a fusionnucleic acid encoding a fusion protein comprising a granule specificprotein and a detectable molecule. This is generally done as is known inthe art, and will depend on the cell type. Generally, for mammaliancells, retroviral vectors and methods are preferred.

The fusion proteins are constructed by methods known in the art. Forexample, the nucleic acids encoding the granule specific protein isligated with a nucleic acid encoding a detectable molecule. Bydetectable molecule herein is meant a molecule that allows a cell orcompound comprising the detectable molecule to be distinguished from onethat does not contain it, i.e., an epitope, sometimes called an antigenTAG, or a fluorescent molecule. Preferred fluorescent molecules includebut are not limited to GFP, BFP, YFP, enzymes including luciferase andβ-galactosidase. These constructs can be made in such a way so that uponexocytosis an epitope, internal to the granule, is exposed at the cellsurface and can then be detected. The epitope is preferably anydetectable peptide which is not generally found on the cytoplasmicmembrane, although in some instances, if the epitope is one normallyfound on the cells, increases may be detected, although this isgenerally not preferred.

In a preferred embodiment, the cell population containing the fusionprotein or detectable granule specific protein is subjected to exocyticconditions. Optionally, a candidate bioactive agent and/or exocyticinhibitor is included. Preferably, the cells are washed. Fluorescence isdetected on the cells. In general, changes of at least about 5% frombaseline are preferred, with at least about 25% being more preferred, atleast about 50% being particularly preferred and at least about 100%being especially preferred. Generally, baseline in this case meansamount of fluorescence prior to exocytic stimulus.

In the invention herein, the same characteristic of exocytosis isevaluated by multiple parameters which results in reduced background andgreater specificity. In contrast, FACS has been used in the past toevaluate two different or unrelated characteristics at the same timewhich identifies cells having those two characteristics, but does notreduce the background for the combined characteristics. The presentinvention can, however, in addition to the identification of multipleexocytosis properties, be combined with the identification of othercellular parameters, as outlined above.

In a preferred embodiment, the cells are subjected to conditions thatnormally cause exocytosis. Pro-exocytic agents include ionomycin, Ca⁺⁺,ionophores (lonomycin, AZ3187), compound 48/80, substance P, complementC3a/C5a, trypsin, tryptase, insulin, interleukin-3, specific IgE,allergen, anti-IgE, or anti-IgG receptor antibodies. These are providedat concentrations depending on the compound as is known in the art,ranging from 1 picomolar to 10 μM, generally. In some cases, it may bedesirable to combine the cells with agents which inhibit exocytosis.Exocytosis inhibitors include but are not limited to Wortmannin, andGenestein, and others known in the art.

In a preferred embodiment, the methods are used to screen candidatebioactive agents for the ability to modulate exocytosis. The candidatebioactive agents may be combined with the cell population before, duringor after exocytosis is stimulated, preferably before. In some instances,it may be desirable to determine the effect of the candidate bioactiveagent, also referred to as “candidate agents” herein, on the cellwherein exocytosis is not induced or wherein exocytosis is inhibited.The candidate bioactive agent can be added to the cell populationexogenously or can be introduced into the cells as described furtherherein.

In a preferred embodiment, as above for cell cycle assays, a library ofdifferent candidate bioactive agents are used.

As above, the candidate bioactive agents are combined or added to a cellor population of cells; again, as outlined above, preferred embodimentsutilize nucleic acid candidate agents and fusion partners; andpreferably retroviral constructs.

Wherein the candidate agents are nucleic acids, methods known in the artsuch as calcium phosphate, electroporation, and injection may be used tointroduce these to the cells. The exocytic stimulus is generallycombined with the cells under physiological conditions. Incubations maybe performed at any temperature which facilitates optimal activity,typically between 4 and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapid highthrough put screening.

As above, a variety of other reagents may be included in the assays, andthe cells are sorted as above. The sorting results in a population ofcells having the desired exocytic properties. In a preferred embodiment,the parameters are set to identify at least one candidate bioactiveagent that modulates exocytosis.

In a preferred embodiment, the bioactive agent is characterized. Thiswill proceed as will be appreciated by those in the art, and generallyincludes an analysis of the structure, identity, binding affinity andfunction of the agent. Generally, once identified, the bioactive agentis resynthesized and combined with the target cell to verify theexocytosis modulation under various conditions and in the presence orabsence of other various agents. The bioactive can be prepared in atherapeutically effective amount to modulate exocytosis and combinedwith a suitable pharmaceutical carrier.

In a preferred embodiment, the cell populations can be subjected tovarious experimental conditions, with and without the candidate agents,and with and without exocytic stimulation or inhibition. Changes inconditions include but are not limited to changes in pH, temperature,buffer or salt concentration, etc. In a preferred embodiment, the pH ischanged, generally by increasing or decreasing the pH, usually by fromabout 0.5 to about 3 pH units. Alternatively, the temperature isaltered, with increases or decreases of from about 5° C. to about 30° C.being preferred. Similarly, the salt concentration may be modified, withincreases or decreases of from about 0.1 M to about 2 M being preferred.

In a preferred embodiment, the cellular phenotype to be modulated issmall molecule (or other candidate agent) toxicity. These are generallyas outlined above for cell viability assays. Small molecule doseresponses can also be compared by comparing the cells with the greatestfunctional response, and then backgating to see if there is more or lesstoxicity associated with those cells.

In a preferred embodiment, the cellular phenotype involves theexpression or activity of cell surface receptors: up to sixteen cellsurface markers may be followed simultaneously, with up to eight beingpreferred. The presence or absence of any particular cell surface markercan be detected by directly and indirectly conjugated antibodies againstany cell surface protein whose cell surface expression reflects animportant functional parameter associated with the cells being studied.The effect of candidate agents such as small molecules can then betested against individual or multiple markers.

In a preferred embodiment, the cellular phenotype involves theexpression or activity of enzymes such as fluorescent based reportersystems that can reporta biological event that occurs simultaneouslywith the primary measurement or is a result of the primary measurement.This reporter system can be a readout of upstream signal transductionpathways that are active in the cytoplasm, or of nucleoartranscriptional or translational events, as well as export events fromthe nucleus or the cell.

In a preferred embodiment, the cellular phenotype involvesprotein-protein interactions (or interactions between other bindingligands), such as dimerization, that can be either disrupted orinstigated by a candidate agent. These events may be measured by theappearance or disappearance of FRET between two labeled binding ligands.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are expressly incorporated by reference intheir entirety.

Example 1 Cell Cycle Assays Using p21 as a Positive Control

Materials and Methods:

Vector Construction:

The coding region of the p21 gene was cloned from Jurkat cDNA by PCRwith an upstream primer covering the start methionine (5′-GATCGGATCCACCACCATGGGCTCAGAACCGGCTGGGGATGTC) and C-terminus (5′-GATCCCAATTTAATGGTTTTATTTGTCATCGTCATCCTTGTAGTCGGGCTTCCTCTTGGAGAAGATCAGCCGGCGTTTG). The single PCR product was directionally cloned into theCRU5-GFP retroviral vector (Rigel, Inc.) through flanking BstXI siteswithin the primers. The resultant construct, CRU5-GFP-p21F (FIG. 1),encodes the GFP fused (in frame) to the human p21 protein with a Glyinsertion at position 2 and a FLAG-epitope at the C-terminus. TheC-terminal 24 amino-acids of p21 were cloned into the CRU5-GFPretroviral vector (Rigel, Inc.) through flanking BstXI sites within thePCR primers:5′GATCCCACCACCATGGGCAAACGGCGGCAGACCAGCATGACAGATTTCTACCACTCCAAACGCCGGCTGATCTTCTCCAA;5′GATCCCAATTTAAATGGTTTTATTTGTCATCGTCATCCTTGTAGTCGGGCTTCCTCTTGGAGAAGATCAGCCGGCGTTTG. The resultant construct, CRU5-GFPp21C (FIG. 1), encodesGFP fused in-frame to KRRQTSMTDFYHSRRLIFSKRKP and a FLAG-epitope at theC-terminus. The C-terminal 24 amin acids of p21, with three alaninemutations, were cloned into the CRU5-GFP retroviral vector (Rigel, Inc.)through flanking BstXI sites within the PCT primers:5′ATCGGATCCACCACCATGGGCAAACGGCGGCAGACCAGCGCCACAGCTGCCTACCACTCC;5′GATCCCAATTTAATGGTTTTATTTGTCATCGTCATCCTTGTAGTCGGGCTTCCTCTTGGAGAAGATCAGCCGGCGTTTG. The resultant construct, CRU5-GFPp21 Cmut (FIG. 1),encodes GFP fused in-frame to KRRQTSATAAYHSRRLIFSKRKP (mutations areunderlined) and a FLAG-epitope at the C-terminus.

Retroviral Transduction:

Phoenix E cells were plated in 6-well plates at 10⁶ cells in 1.5 mlcomplete-DMEM (DMEM+10% FBS+Pen/Strep) and incubated at 37° C. for 16hours. CaCl₂-precipitation transfection was performed (2 μl DNA (1μg/μl), 30.5 μl 2M CaCl₂, 217.5 μl H₂O, 0.5 ml 2×HBS) with theCRU5-IRES-GFP vector or CRU5-p21F-IRES-GFP clone in the presence of 50μM chloroquine for 8 hours at 37° C. The transfection-medium was removedand replaced with 2 ml complete-DMEM and the cells were furtherincubated for 16 hours at 37° C. The medium was changed to 1.5 mlcomplete-RPMI (RPMI+10% FBS+Pen/Strep) and incubated at 32° C. for 48hours. The virus supernatant from transfected plates was filtered (0.45μm) and transferred to a 6-well plate. An 100 μl aliquot (5×10⁶ cells)of Jurkat T-cells expressing the ecotrophic receptor (JurkatE) was addedto each well. Polybrene was added to a final concentration of 5 μg/ml.The plates were sealed with parafilm and centrifuged at 32° C. for 90minutes at 2500 RPM. The parafilm was removed and the plate incubatedovernight at 37° C. The medium was changed after 16 hours to 4 mlcomplete-RPMI and incubated at 37° C. for 72 hours.

Cell Cycle FACS-Assay:

The retroviral vector-transduced cells were pelleted and resuspended at10⁶ cells/ml in complete-RPMI. One volume (1 ml) of 4 μM PKH26 celltracking dye (Sigma) was added to the cells and incubated at 25° C. for5 minutes. The suspension was diluted 5-fold and the cells pelleted at400×g for 10 minutes at 25° C. The cells were further washed twice with6 ml complete-RPMI and incubated at 3×10⁵ cells/ml in a 6-well plate for72 hours. The labeled cells were pelleted and resuspened at 10⁶ cells/mlin complete-RPMI containing 5 ug/ml Hoechst 33342 (Molecular Probes) andincubated at 37° C. for 2 hours. The stained cells were pelleted andresuspened at >10⁶ cells/ml in FACS buffer (PBS/0.5% FCS/5 ug/ml Hoechst33342). The cells were subjected to flow-cytometric analysis on a MoFlocytometer (Cytomation) equipped with three lasers. Forward and sidescatter were triggered with a 488 nm-line argon laser and scatteredlight was collected with a forward scatter detector and 488 nm band passfilter. GFP was excited with a 488 nm-line argon laser and emitted lightwas collected through a 530 nm-band pass filter. PKH26 cell tracking dyewas excited with a 533 nm-line HeNe-laser and emitted light wascollected through a 570 nm-band pass filter. Hoechst 33342 dye wasexcited with a UV-laser and emitted light was collected through a 450nm-band pass filter.

Results:

Jurkat T-cells were transduced with retroviral vectors encoding humanp21 (Gp21), or the PCNA binding C-terminal 24 amino acids (Gp21C) fusedto GFP (FIG. 1). A non-PCNA binding mutant version of the p21 C-terminal24 amino acids (Gp21 Cmut, Cayrol et al., Oncogene 16:311 (1998)) servedas a negative control. Expression of the transduced p21 could bedistinguished from the endogenous protein by the FLAG-epitope by Westernblotting (not shown). Expression of the fusion proteins was reported inthe FACS by GFP fluorescence (FIG. 2B). Transduced cells were pulsedlabeled with a cell tracking compound, pkh26, which incorporates redfluorescent aliphatic molecules into the cell membrane by selectivepartioning, allowing a correlation between cell cycling and fluorescentintensity: arrested cells remain cell tracker dye bright; cycling cellsdilute the signal and dim. As shown in FIG. 2C, live GFP-p21-expressingcells gated on GFP, demonstrated a higher red fluorescence than vectortransduced cells expressing identical GFP levels, indicating cell cyclearrests. A similar effect was seen in the Gp21C expressing cells,however, Gp21-Cmut was identical to non-expressing cells. The DNAcontent of the same GFP-gated cells is shown in FIG. 2D. Gp21 expressingcells are arrested in the G1 phase of the cell cycle, Gp21C-expressingcells show G1 and G2 checkpoint accumulation, consistent with previousresults (Wade Harper, et al., 1993; Cayrol et al., 1998). The Gp21 Cmutexpressing cells show a normal cell cycle distribution. Viable,arrested, expressing cells (satisfying the three initial parameters)were sorted based on DNA content into separate chambers: leftdeflection, G1; right deflection, G2.

Example 2 Population Based Exocytic Enzyme Activity Measurements

Materials:

All chemicals were obtained from Sigma Chemical Co. Dyes and glucuronidewere obtained from Molecular Probes, Inc. Cell lines MC-9 and RBL-2H3were obtained from American Type Culture Collection (ATCC). Cell culturereagents were obtained from Fisher Scientific and molecular biologyreagents from Clontech Inc.

Cell Culture:

MC-9 cells were maintained as suspension cultures in flasks in mediaconsisting of DMEM with L-arginine (116 mg/ml), L-asparagine (36 mg/ml),sodium pyruvate (1 mM), non-essential amino acids (0.1 mM), folic acid(6 mg/ml), 2-mercaptoethanol (0.05 mM), L-glutamine (2 mM), heatinactivated fetal bovine serum (10%), and 10% T-stim conditioned media(Collaborative Research, Inc.). The cells were kept at a density ofbetween 0.25 and 2×10⁶/ml. Experiments were only conducted on cellswhich were greater than 95% viable as determined by trypan blueexclusion. RBL-2H3 cells were maintained as adherent cultures onuncoated (tissue culture treated) flasks in media consisting of EaglesMEM with 2 mM L-Glutamine and Earl's BSS, 15% heat inactivated fetalbovine serum. The cells were passaged (0.05% trypsin) so that they werenot confluent for more than one day.

Exocytosis Stimulation Protocol:

Experiments were carried out in modified tyrodes buffer (MT) whichconsisted of NaCl (137 mM), KCl (2.7 mM), CaCl₂ (1.8 mM), MgCl₂ (1 mM),Glucose (5.6 mM), Hepes (20 mM, pH 7.4), and bovine serum albumin(0.1%). MC-9 cells were spun at 400×g and the media was aspirated. Thecells were then washed with MT, respun/aspirated and taken up in MT at adensity of 5×10⁶ cells/ml. Cells were then treated with either DMSO orionophore for 30 minutes (or the time was varied if a timecourse). Thecells were then pelleted with the supernatant collected for enzymaticanalysis; in some cases, the cells then processed for flow cytometry.All stimulations were carried out at 37′C. Stimulations of RBL-2H3 cellswere carried out by washing the adherent cells one time in MT and thenadding warmed MT (1 ml/10⁶ cells) containing the stimulus. The cellswere incubated at 37° C. for 30 minutes and the supernatant washarvested for further analysis. In some of the examples, (Examples 4-6),the plate bound cells were stained for annexin and then removed from theflask using No-Zyme (Collaborative Research, Inc.) for furtherprocessing for flow cytometry. For stimulation of RBL-2H3 cells withantigen crosslinking the cells were incubated overnight with IgEanti-DNP (Sigma Chemical Co.) in complete media at a concentration of 50ng/ml. The following day they were washed one time in MT and stimulatedas described above with the exception that bovine serum albumin coupledto DNP was used as the stimulus at 100 ng/ml.

Population Based Enzyme Assays:

Enzyme assays were carried out on cell supernatants and pelletsfollowing exocytic stimulation. Cell supernatants were harvested afterstimulation, chilled on ice, and the post 5000×g spin supernatant wascollected for enzyme activity analysis. Similarly, cell pellets werecollected/lysed in MT containing 0.1% triton X-100 and the post 5000×gspin supernatant was collected for enzyme activity analysis. For eachanalysis 100 μl of lysate or supernatant was mixed with 100 μl ofreaction buffer (40 mM Citrate, pH 4.5) containing 2 mM substrate(4-methylumbelliferyl β-D Glucuronide [glucuronidase substrate] or4-methylumbelliferyl N-acetyl β-D glucosaminide [hexosaminidasesubstrate]) in a solid black 96 well plate (Costar, Inc.) and incubatedat 37° C. for 15 minutes. The plate was read on a fluorescence platereader (Wallac, Inc.) using excitation 380 nm/emission 440 nm filtersevery 3 minutes for five times to obtain an enzymatic rate; analyseswere carried out in triplicate.

Flow Cytometry:

Cells processed for stimulation and staining were taken up in MT on iceand filtered through a 100 μm filter prior to cytometry. Cells wereanalyzed using a FACSCAN (Becton Dickinson Inc., laser line 458 nm) or aMo-Flo (Cytomation, Inc., laser lines 350 nM broadband (UV), 488 nm, and647 nm) Cytometer. Cells were sorted, if desired using the Mo-Flo.

Results:

The results are shown in FIG. 4. Enzymatic activity in the cellsupernatant was measured for MC9 (A) and RBL 2H3 (B) cells under variousconditions. A) MC-9 cells were stimulated in the presence of DMSO (−) or2 μm lonomycin (+) for 30 minutes. The supernatant was collected andanalyzed for glucuronidase or hexosaminidase activity. Stimulatedrelease of granule enzymatic activity is evident. B) RBL-2H3 cells weresensitized for 16 hours with varying amounts of IgE anti-DNP andstimulated to exocytose by exposure to increasing amounts of the antigenBSA-DNP. A dose response of both antibody and antigen is evident in themeasured supernatant hexosaminidase activity.

Example 3 Mast Cell Exocytic Light Scatter Changes

The cells were prepared as described in Example 2, and light scatterproperties were determined.

Results:

The results are shown in FIG. 4. Light scatter changes observed on theflow cytometer (side scatter vs. forward scatter) are plotted asbivariate histograms for RBL-2H3 cells (A, D) and MC-9 cells (B, C, E,F). Cells were stimulated with the ionophore A23187 (0.5 ug/ml) andobserved at various timepoints [0 minutes (A, C), 5 minutes (E), 10minutes (D), and 30 minutes (B, F)]. Time dependent scatter changes areevident in both cell lines with significant changes occurring during thefirst 10 minutes which represents the major bolus of exocytosis in thesecells.

Example 4 Styryl Dyes Detect Mast Cell Exocytosis by FACS

Styrl Dye Staining:

The cells were prepared as described above. Styryl dyes (FM1-43 orFM4-64; Molecular Probes, Inc.) were diluted to a final concentration of250 nM in MT and were incorporated into the stimulation buffer (seeExample 2). After the stimulation protocol the cells were spun down,aspirated and resuspended in fresh ice cold MT. The cells were thenready for analysis in the flow cytometer (see Example 2).

Results:

The results are shown in FIG. 6. MC-9 cells were stimulated (blue=DMSO,red=2 μM ionomycin) in the presence either FM 4-64 (A, B) or FM 1-43 (C,D, E). A) FM 4-64 labeled cells detected in the flow cytometer influorescence channel 1. B) FM 4-64 labeled cells detected in the flowcytometer in fluorescence channel 3. C) FM 1-43 labeled cells detectedin the flow cytometer in fluorescence channel 1. D) FM 1-43 labeledcells detected in the flow cytometer in fluorescence channel 3. There isa clear stimulation dependent increase of fluoreceence intensity withboth dyes; FM 4-64 being the most red-shifted and predominantly detectedin channel 3 while FM 1-43 is more broadly fluorescent being detected inboth channels 1 and 3. E) MC-9 cells were preincubated with varyingdoses of the PI-3 kinase inhibitor wortmannin (1 μM-bar1, 100 nM-bar2,10 nM-bar3, and 0 nM-bars 1 and 2) prior to stimulation with A23187 (0.5ug/ml, bars 1-4) or DMSO (bar 5) in the presence of FM 1-43. The meanchannel shift detected in the flow cytometer in fluorescence channel 1is plotted as a bar graph. Wortmannin, a known inhibitor of mast cellexocytosis, causes a dose dependent decrease in the FM 1-43 signalindicating that FM 1-43 signal reflects the degree of degranulation inthe MC-9 mast cell line.

Example 5 Annexin-V Staining Detects Mast Cell Exocytosis by FACS

Materials:

Annexin-V biotin, Annexin-V FITC and streptavidin APC were obtained fromCaltag Laboratories. Other materials and methods used herein can beincorporated from the other examples, particularly Example 2.

Annexin-V Staining:

Cells post exocytic stimulus were stained with annexin-PITC at adilution of 11100 in MT for 10 minutes at room temperature. The cellswere then washed one time in MT, taken up in MT and viewed in the flowcytometer or microscope. For indirect labeling, annexin-biotin was addedto the MT during the stimulation procedure at a dilution of 1/200. Thecells were then pelleted in ice cold MT, spun, aspirated, and taken upin ice cold MT with streptavidin-APC at a dilution of 1/200 and kept onice for 15 minutes. After pelieting the cells and aspirating theStreptavidin-APC, the cells were resuspended in MT and viewed in theflow cytometer. In some experiments different secondaries were appliedsuch as streptavidin alexa 488 or 594 (Molecular Probes, Inc.) forvisualization in the microscope.

Results:

The results are shown in FIG. 7. MC-9 cells were stimulated with eitherDMSO (Figures A and B) or 2 μm ionomycin (Figures C and D) and thenstained with both propidium iodide [α](Figures A and C) andannexin-V-FITC (Figures B and D). Stimulation with this dose ofionomycin does not compromise the plasma membrane as demonstrated by nosignificant increase in PI staining in the exocytosing cells.Degranulation results in a significant increase in annexin binding asseen comparing Figures D and B.

Example 6 Annexin-V-FITC Stains Exocytic Granules in MC-9 CellsVisualized by Confocal Microscopy

Microscopy:

Cells post stimulation or staining were mounted onto glass slides andcoverslipped; these were directly visualized by brighffield andfluorescence microscopy on an inverted microscope (TE300, Nikon) usingstandard BFP, FITC, or TRITC filter sets. Some images were obtainadusing an inverted confocal scanning microscope (Zeiss, Inc., Bio-Rad,Inc.) using standard FITC and TRITC filter sets.

Results:

MC-9 cells were stimulated with 2 μm ionomycin or DMSO and stained withannexin-V-FITC and mounted for confocal microscopy (data not shown). Allviable unstimulated cells show low annexin binding and no cell surfacegranular staining.

Example 7 Annexin-V-FITC Stains Exocytic Granules in RBL-2H₃CellsStimulated with Antigen Crosslinking

Except as othewise stated below, the methods for this example aredescribed in the preceding examples.

Results:

RBL-2H3 cells were sensitized with IgE and stimulated to exocytosa witheither MT buffer only or BSA-DNP antigen (100 ng/ml) and then stainedwith annexin-V-FITC. Numerous cell surface granules are stained in theantigen stimulated cells similar to the pattern seen in MC 9-cells.Viable unstimulated cells show negligible annexin-V staining (data notshown).

Example 8 In Situ Enzymology of Exocytosing Cells Visualized in the FACS

In Situ Enzymology:

MC-9 cells were stimulated for exocytosis as described above and thenincubated in enyme substrate buffer (BSA free MT, pH 4.3-7.4 range)containing the substrate ELF-97 Glucuronide (250 μM) for 15 minutes at37° C. The cells were washed one time in MT and then viewed in themicroscope or on the flowcytometer. Further methodologies are describedin the preceding examples.

Results:

The results are shown in FIG. 8. MC-9 cells ware stimulated with DMSO(Figure A) or A23187 (0.5 μg/ml—Figure B and C) and then stained for insitu glucuronidase activty. A) Flow cytometer histogram of ELF-97detection indicative of cell surface enzymatic precipitate. Very lowsignal is seen in the DMSO treated cells. B) Flow cytometer histogram ofELF-97 detection indicative of cell surface enzymatic precipitate. Asignificant increase in signal is seen upon secretory stimulation withionophore. C) pH profile of the cell surface enzymatic activity. MTbuffer, prepared at different pHs, was used to pH profile the signalseen in the flow cytometer. The bar graphs represent the percentage ofmaximal signal (as measured by mean channel shift in the flow cytomoter)observed. The enzymatic activity is pH dependent with a peak at lessthan pH 6; this is consistent with enzymatic acitivity derived from anacidic secretory granule.

Example 9 Lysotracker Green is Released from Mast Cell Granules UponExocytosis and can be Detected by FACS

Lysotracker Dye Staining:

Lysotracker dyes (blue, green, and red) were loaded into cells bydiluting them to a final concentration of 1 μM in complete media andincubating the cells for 60 minutes at 37″C in their presence. Afterloading, the cells were washed two times in MT and then were ready forfurther analysis or stimulation. Further methodologies are described inthe preceding examples.

Results:

The results are shown in FIG. 9. MC-9 cells were loaded with Lysotrackergreen for 1 hour and then stimulated with either DMSO or ionomycin (2μM) and viewed in the flow cytometer. Shown is a histogram offluorescence intensity detected in channel 1; a significant loss ofsignal is seen in the ionophore stimulated sample as compared to theDMSO control which is reflective of the release of lysotracker green dyefrom the secretory granules.

Example 10 Multiparameter Analysis—Lysotracker Green, Annexin-V-APC,Forward and Side Scatter

Except as otherwise described below, the methodologies described in thepreceding examples were used.

Results:

The results are shown in FIG. 10. MC-9 cells were treated with differentdoses of ionomycin (0 μM-A, E; 1 μM-B, F; 2 μM-C, G; and 3 μM-D, H) andobserved in the flow cytometer with four parameters simultaneously. Thecells were loaded with lysotracker green for one hour and thenstimulated and stained for annexin-VAPC. Figures A-D: Bivariatehistograms of side vs. forward light scatter. Note the dose dependentchanges in both parameters from left to right as forward scatterincreases and side scatter decreases. Figures E-H: Bivariate histogramsof annexin-V-APC vs. Lysotracker green signals. As exocytosis increases(left to right) annexin signal becomes greater as the lysotracker signaldecreases. This reflects the binding of annexin-V to the cell surfacegranules and the loss of lysotracker from these granules as they areexposed to the extracellular milieu.

Example 11 Multiparameter Analysis—FM 1-43, Annexin-V-APC, Forward andSide Scatter

Except as otherwise noted below, the methodologies described above wereused herein.

Results:

MC-9 cells were treated with either DMSO or ionomycin (2 μM) andobserved in the flow cytometer with four parameters simultaneously. Thecells were stimulated in the presence of FM 1-43 and stained forannexin-V-APC (data not shown). There were stimulation dependent changesin both parameters at the 30 minute timepoint. There were stimulationdependent increases in both signals. The changes reflect the binding ofannexin-V to the cell surface granules and the simultaneous coupledendocytosis of the FM 1-43 dye into the MC 9 cells.

Example 12 Simultaneous Multiparameter Measurements in the FAGSCorrelate with Population Based Enzyme Readouts

Calcium Signaling Assays:

MC-9 or RBL-2H3 cells were washed one time in MT and loaded with theCa⁺⁺ sensitive probe Fluo-3 (1 μM, Molecular Probes, Inc.) in MT at 37°C. for 20 minutes. The cells were washed one time in warm MT and thenstimulated using the protocol described above. The signal due to rise inthe intracellular Ca⁺⁺ concentration was visualized using either theflow cytometer (see below), fluorescence microscopy, or read on afluorescence plate reader (Wallac, Inc.). Loading of the cells wasdetermined by releasing the intracellular dye with MT containing 0.1%triton X-100.

Except as otherwise noted below, the methodologies described above wereused herein.

Results:

The results are shown in FIG. 11. MC-9 cells were stimulated in thepresence of FM 1-43 and annexin-V-APC stained as described in themethods above. At various timepoints after ionomycin stimulation thecells were put on ice and either analyzed by flow cytometry or forenzymatic activity (cell supernatant). The parameters forward scatter,FM 1-43, annexin-V-APC, and hexosaminidase are plotted on the graphrelative to the maximal response for each parameter. For calciumsignaling, a separate tube of cells was loaded with Fluo-3 and underwentthe identical procedure. The timecourses of the cytometry basedparameters indicate that they correlate quite well with exocytosis asmeasured by hexosaminidase release. Forward scatter, in this example,shows an effect which varies both positively and negatively with time.

Example 12 Expression of VAMP-GFP and VAMP-FRET Constructs

cDNA Constructs:

VAMP-GFP construct:

The rat VAMP-2 cDNA (obtained from R. Scheller, Stanford University) wasPCR modified to introduce: (1) a 5′ BstXI site encoding a concensusKozak and glycine insertion (a.a.2) to facilitate expression and in vivostability, respectively; (2) a serine-glycine linker with a BamHI siteat the 3′ end. The GFP coding sequence from CdimGFP (Clontech, Inc.) wasPCR modified to introduce a 3′ BstXI site encoding a stop codon. TheVAMP-GFP fusion was constructed by ligating the modified rVAMP and GFPPCR fragments through a common BamHI site in the serine-glycine linkerto create an in-frame fusion protein with the following sequence:

MGSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKM MIILGVICAIILIIIIVYFSTGSGSGSGSGSGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKZ

The VAMP sequence is underlined, the serine-glycine linker is italicizedand the GFP sequence is in regular text.

The VAMP-GFP fusion sequence was cloned into the 96.7 retroviral vectorwith directional BstXI sites to create pVG. The sequence was verified bysequencing in both directions. Proper expression was verified intransfected and infected cells by Western analysis and fluorescencemicroscopy.

Trp-FRET Construct:

The GFP coding sequence from cGFP (Clontech, Inc.) was PCR modified tocreate: (1) a 5′ BstXI site encoding a concensus Kozak and glycineinsertion (a.a.2) to faciltate expression and in vivo stability,respectively; (2) a 3′-end SacII site encoding Ala228 at the C-terminus.The BFP coding sequence from cBFP (Clontech, Inc.) was PCR modified tocreate: (1) a 5′ BamHI site encoding Ser2; (2) a 3′-end BstXI encoding astop codon. A SacII-BamHI conversion linker encoding Factor X andtryptase protease cleavage sites, flanked by GSGS spacers(GSGSIEGRLRKQGSCS) was used to fuse the GFP and BFP to create anin-frame fusion protein with the following sequence:

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAA GSGSIEGRLRKQGSGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKZ

The GFP sequence is underlined, the Factor X/tryptase site linker isitalicized and the BEP sequence is in regular text.

The VAMP-GFP fusion sequence was cloned into the BamHI and BstXI sitesof the retroviral vector 96.7 to create pGX/TB. The sequence wasverified by sequencing in both directions. Proper expression wasverified in transfected and infected cells by Western analysis andfluorescence microscopy.

The VAMP-GFP encoding sequence was PCR modified to create a 3′-end SacIIsite encoding Ala228 at the C-terminus. This fragment was cleaved withXhoI and SacII and cloned into the XhoI/SacII sites of pGX/TB to createpVGXITB (Trp-FRET), encoding the rVAMP-2-BFP-Factor X/Tryptase sites-GFPfusion protein with the following sequence:

GSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWVVKNLKM MIILGVICAIILIIIIVYFSTGSGSGSGSGSGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGSGSIEGRRKLQGSGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKIFCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKZ

The VAMP sequence is underlined, the serine-glycine linker isitalicized, the Factor X/tryptase site linker is in bold and the GFP andBFP sequences are in shown in regular text.

The rVAMP-2-BFP-Factor X/Tryptase sites-GFP fusion sequence was verifiedby sequencing in both directions. Proper expression was verified intransfected and infected cells by Western and fluorescence microscopyand FACS analysis.

Transfections and Infections:

To infect MC-9 and RBL-2H3 cells with recombinant retrovirusesexpressing the Vamp constructs the following procedure was carried outPhoenix E or A cells (obtained from G. Nolan, Stanford Univ.) wereplated out in 6 well plates at 8×10E5 cells in 1.5 ml media (DMEM, 10%FBS) on day one. On day two 5 μg of DNA was transfected into the cellsusing the CaPO₄ precipitation method in the presence of 50 μMchloroquine. The precipitate was incubated with the cells for 8 hours at37° C. at which time the medium was removed, washed once with freshmedia and replaced with either fresh MC-9 or RBL-2H3 media; the cellswere then incubated at 32° C. for 48-72 hours. The supernatant from thePhoenix cells (viral supernatant) was spun at 1000×g for 10 minutes andprotamine sulfate was added to a final concentration of 5 μg/ml; thissupernatant was added to the MC-9 or RBL-2HS freshly trypsinized) cellsin a 6 well plate (5×10E5 cells per well) and the mixture was spun at1000×g for 90 minutes at room temperature The cells were then incubatedat 32° C. for 16 hours. The viral supernatant was removed and freshmedia was added; target gene expression was seen after 24 hours postinfection.

We claim:
 1. A method of screening for a bioactive agent capable ofaltering a cellular phenotype, said method comprising: a) combining atleast one candidate bioactive agent and a population of cells; and b)sorting said cells in a FACS machine by separating said cells on thebasis of at least five cellular parameters.
 2. A method according toclaim 1 wherein a library of candidate bioactive agents are combinedwith said population.
 3. A method of screening for a bioactive agentcapable of altering a cellular phenotype, said method comprising: a)introducing a library of nucleic acids each encoding a candidatebioactive agent into a population of cells; and b) sorting said cells ina FACS machine by separating said cells on the basis of at least threecellular parameters.
 4. A method according to claim 3 wherein saidlibrary is a retroviral library.
 5. A method according to claim 3 or 4wherein said cellular phenotype is exocytosis and said cellularparameters are selected from the group consisting of light scattering,fluorescent dye uptake, fluorescent dye release, annexin granulebinding, surface granule enzyme activity, and the quantity of granulespecific proteins.
 6. A method according to claim 5 further comprisingsubjecting said cells to conditions that normally cause exocytosis.
 7. Amethod according to claim 3 or 4 wherein said cellular phenotype is cellcycle regulation and said cellular parameters comprise cell viability,proliferation, and cell phase.
 8. A method according to claim 3, 4, 5, 6or 7 wherein said nucleic acids comprise fusion nucleic acidscomprising: a) said nucleic acid encoding said candidate bioactiveagents; and b) a detectable moiety.
 9. A method according to claim 1, 2,3, 4, 5, 6, 7 or 8 wherein said cells are tumor cells.
 10. A methodaccording to claim 8 wherein said detectable moiety is a fluorescentprotein.